Alpha-amylase mutants with altered properties

ABSTRACT

The present invention relates to variants (mutants) of parent Termamyl-like alpha-amylases, which variant has alpha-amylase activity and exhibits altered stability, in particular at high temperatures and/or at low pH relative, and/or low Ca2+ to the parent alpha-amylase.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims, under 35 U.S.C. 119, priority of Danishapplication no. PA 2000 01160, filed Aug. 1, 2000, application no. PA2000 01354, filed Sep. 12, 2000, application no. PA 2000 01687, filedNov. 10, 2000, and application no. PA 2001 00655, filed on Apr. 26,2001, and this application claims the benefit of U.S. provisionalapplication No. 60/225140, filed on Aug. 14, 2000, application No.60/233986, filed Sep. 30, 2000, application No. 60/24104, filed Nov. 16,2000, application No. 60/286869, filed Apr. 26, 2001, the contents ofwhich are fully incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to variants (mutants) of parentTermamyl-like alpha-amylases, which variant has alpha-amylase activityand exhibits an alteration in at least one of the following propertiesrelative to said parent alpha-amylase: stability under, e.g., hightemperature and/or low pH conditions, in particular at low calciumconcentrations. The variant of the invention are suitable for starchconversion, ethanol production, laundry wash, dish wash, hard surfacecleaning, textile desizing, and/or sweetner production.

BACKGROUND OF THE INVENTION

[0003] Alpha-Amylases (alpha-1,4-glucan-4-glucanohydrolases, E.C.3.2.1.1) constitute a group of enzymes, which catalyze hydrolysis ofstarch and other linear and branched 1,4-glucosidic oligo- andpolysaccharides.

BRIEF DISCLOSURE OF THE INVENTION

[0004] The object of the present invention is to provide Termamyl-likeamylases which variants in comparison to the corresponding parentalpha-amylase, i.e., un-mutated alpha-amylase, has alpha-amylaseactivity and exhibits an alteration in at least one of the followingproperties relative to said parent alpha-amylase: stability under, e.g.,high temperature and/or low pH conditions, in particular at low calciumconcentrations.

[0005] Nomenclature

[0006] In the present description and claims, the conventionalone-letter and three-letter codes for amino acid residues are used. Forease of reference, alpha-amylase variants of the invention are describedby use of the following nomenclature:

[0007] Original amino acid(s): position(s): substituted amino acid(s)

[0008] According to this nomenclature, for instance the substitution ofalanine for asparagine in position 30 is shown as:

[0009] Ala30Asn or A30N

[0010] a deletion of alanine in the same position is shown as:

[0011] Ala30* or A30*

[0012] and insertion of an additional amino acid residue, such aslysine, is shown as:

[0013] Ala30AlaLys or A30AK

[0014] A deletion of a consecutive stretch of amino acid residues, suchas amino acid residues 30-33, is indicated as (30-33)* or Δ(A30-N33).

[0015] Where a specific alpha-amylase contains a “deletion” incomparison with other alpha-amylases and an insertion is made in such aposition this is indicated as:

[0016] *36Asp or *36D

[0017] for insertion of an aspartic acid in position 36. Multiplemutations are separated by plus signs, i.e.:

[0018] Ala30Asp+Glu34Ser or A30N+E34S representing mutations inpositions 30 and 34 substituting alanine and glutamic acid forasparagine and serine, respectively.

[0019] When one or more alternative amino acid residues may be insertedin a given position it is indicated as A30N,E or

[0020] A30N or A30E

[0021] Furthermore, when a position suitable for modification isidentified herein without any specific modification being suggested, itis to be understood that any amino acid residue may be substituted forthe amino acid residue present in the position. Thus, for instance, whena modification of an alanine in position 30 is mentioned, but notspecified, it is to be understood that the alanine may be deleted orsubstituted for any other amino acid, i.e., any one of:

[0022] R,N,D,A,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V.

[0023] Further, “A30X” means any one of the following substitutions:

[0024] A30R, A30N, A30D, A30C, A30Q, A30E, A30G, A30H, A30I, A30L, A30K,A30M, A30F, A30P, A30S, A30T, A30W, A30Y, or A30V; or in short:A30R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V.

[0025] If the parent enzyme—used for the numbering—already has the aminoacid residue in question suggested for substitution in that position thefollowing nomenclature is used:

[0026] “X30N” or “X30N,V” in the case where for instance one or N or Vis present in the wildtype. Thus, it means that other correspondingparent enzymes are substituted to an “Asn” or “Val” in position 30.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027]FIG. 1 is an alignment of the amino acid sequences of five parentTermamyl-like alpha-amylases. The numbers on the extreme left designatethe respective amino acid sequences as follows:

[0028] 1: SEQ ID NO: 4 (SP722)

[0029] 2: SEQ ID NO: 2 (SP690)

[0030] 3: SEQ ID NO: 10 (BAN)

[0031] 4: SEQ ID NO: 8 (BLA)

[0032] 5: SEQ ID NO: 6 (BSG).

DETAILED DISCLOSURE OF THE INVENTION

[0033] The object of the present invention is to provide Termamyl-likeamylases, which variants have alpha-amylase activity and exhibitsaltered stability at high temperatures and/or at low pH, in particularat low calcium concentrations.

[0034] Termamyl-like Alpha-amylases

[0035] A number of alpha-amylases produced by Bacillus spp. are highlyhomologous (identical) on the amino acid level. The identity of a numberof known Bacillus alpha-amylases can be found in the below Table 1:TABLE 1 Percent identity 707 AP1378 BAN BSG SP690 SP722 AA560 Termamyl707 100.0 86.4 66.9 66.5 87.6 86.2 95.5 68.1 AP1378 86.4 100.0 67.1 68.195.1 86.6 86.0 69.4 BAN 66.9 67.1 100.0 65.6 67.1 68.8 66.9 80.7 BSG66.5 68.1 65.6 100.0 67.9 67.1 66.3 65.4 SP690 87.6 95.1 67.1 67.9 100.087.2 87.0 69.2 SP722 86.2 86.6 68.8 67.1 87.2 100.0 86.8 70.8 AA560 95.586.0 66.9 66.3 87.0 86.8 100.0 68.3 Termamyl 68.1 69.4 80.7 65.4 69.270.8 68.3 100.0

[0036] For instance, the B. licheniformis alpha-amylase comprising theamino acid sequence shown in SEQ ID NO: 8 (commercially available asTermamyl™) has been found to be about 81% homologous with the B.amyloliquefaciens alpha-amylase comprising the amino acid sequence shownin SEQ ID NO: 10 and about 65% homologous with the B. stearothermophilusalpha-amylase (BSG) comprising the amino acid sequence shown in SEQ IDNO: 6. Further homologous alpha-amylases include SP690 and SP722disclosed in WO 95/26397 and further depicted in SEQ ID NO: 2 and SEQ IDNO: 4, respectively, herein. Other amylases are the AA560 alpha-amylasederived from Bacillus sp. and shown in SEQ ID NO: 12, and the #707alpha-amylase derived from Bacillus sp., shown in SEQ ID NO: 13 anddescribed by Tsukamoto et al., Biochemical and Biophysical ResearchCommunications, 151 (1988), pp. 25-31.

[0037] The KSM AP1378 alpha-amylase is disclosed in WO 97/00324 (fromKAO Corporation).

[0038] Still further homologous alpha-amylases include the alpha-amylaseproduced by the B. licheniformis strain described in EP 0252666 (ATCC27811), and the alpha-amylases identified in WO 91/00353 and WO94/18314. Other commercial Termamyl-like alpha-amylases are comprised inthe products sold under the following tradenames: Optitherm™ andTakatherm™ (Solvay); Maxamyl™ (available from Gist-brocades/Genencor),Spezym AA™ and Spezyme Delta AA™ (available from Genencor), andKeistase™ (available from Daiwa), Dex lo, GC 521 (available fromGenencor) and Ultraphlow (from Enzyme Biosystems).

[0039] Because of the substantial homology found between thesealpha-amylases, they are considered to belong to the same class ofalpha-amylases, namely the class of “Termamyl-like alpha-amylases”.

[0040] Accordingly, in the present context, the term “Termamyl-like”alpha-amylase” is intended to indicate an alpha-amylase, in particularBacillus alpha-amylase, which, at the amino acid level, exhibits asubstantial identity to Termamyl, i.e., the B. licheniformisalpha-amylase having the amino acid sequence shown in SEQ ID NO: 8,herein.

[0041] In other words, all the following alpha-amylases, which has theamino acid sequences shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12 and 13herein are considered to be “Termamyl-like alpha-amylase”. OtherTermamyl-like alpha-amylases are alpha-amylases i) which displays atleast 60%, such as at least 70%, e.g., at least 75%, or at least 80%, atleast 85%, at least 90%, at least 95%, at least 97%, at least 99%homology (identity) with at least one of said amino acid sequences shownin SEQ ID NOS: 2, 4, 6, 8, 10, 12, and 13, and/or is encoded by a DNAsequence which hybridizes to the DNA sequences encoding theabove-specified alpha-amylases which are apparent from SEQ ID NOS: 1, 3,5, 7, 9, and of the present specification (which encoding sequencesencode the amino acid sequences shown in SEQ ID NOS: 2, 4, 6, 8, 10 and12 herein, respectively).

[0042] Homology

[0043] The homology may be determined as the degree of identity betweenthe two sequences indicating a derivation of the first sequence from thesecond. The homology may suitably be determined by means of computerprograms known in the art such as GAP provided in the GCG programpackage (described above). Thus, Gap GCGv8 may be used with the defaultscoring matrix for identity and the following default parameters: GAPcreation penalty of 5.0 and GAP extension penalty of 0.3, respectivelyfor nucleic acidic sequence comparison, and GAP creation penalty of 3.0and GAP extension penalty of 0.1, respectively, for protein sequencecomparison. GAP uses the method of Needleman and Wunsch, (1970), J.Mol.Biol. 48, p.443-453, to make alignments and to calculate the identity.

[0044] A structural alignment between Termamyl (SEQ ID NO: 8) and, e.g.,another alpha-amylase may be used to identify equivalent/correspondingpositions in other Termamyl-like alpha-amylases. One method of obtainingsaid structural alignment is to use the Pile Up programme from the GCGpackage using default values of gap penalties, i.e., a gap creationpenalty of 3.0 and gap extension penalty of 0.1. Other structuralalignment methods include the hydrophobic cluster analysis (Gaboriaud etal., (1987), FEBS LETTERS 224, pp. 149-155) and reverse threading(Huber, T; Torda, AE, PROTEIN SCIENCE Vol. 7, No. 1 pp. 142-149 (1998).

[0045] Hybridisation

[0046] The oligonucleotide probe used in the characterisation of theTermamyl-like alpha-amylase above may suitably be prepared on the basisof the full or partial nucleotide or amino acid sequence of thealpha-amylase in question.

[0047] Suitable conditions for testing hybridisation involve pre-soakingin 5×SSC and prehybridizing for 1 hour at 40° C. in a solution of 20%formamide, 5×Denhardt's solution, 50 mM sodium phosphate, pH 6.8, and 50mg of denatured sonicated calf thymus DNA, followed by hybridisation inthe same solution supplemented with 100 mM ATP for 18 hours at 40° C.,followed by three times washing of the filter in 2×SSC, 0.2% SDS at 40°C. for 30 minutes (low stringency), preferred at 50° C. (mediumstringency), more preferably at 65° C. (high stringency), even morepreferably at 75° C. (very high stringency). More details about thehybridisation method can be found in Sambrook et al., Molecular Cloning:A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989.

[0048] In the present context, “derived from” is intended not only toindicate an alpha-amylase produced or producible by a strain of theorganism in question, but also an alpha-amylase encoded by a DNAsequence isolated from such strain and produced in a host organismtransformed with said DNA sequence. Finally, the term is intended toindicate an alpha-amylase, which is encoded by a DNA sequence ofsynthetic and/or cDNA origin and which has the identifyingcharacteristics of the alpha-amylase in question. The term is alsointended to indicate that the parent alpha-amylase may be a variant of anaturally occurring alpha-amylase, i.e., a variant, which is the resultof a modification (insertion, substitution, deletion) of one or moreamino acid residues of the naturally occurring alpha-amylase.

[0049] Parent Termamyl-like Alpha-amylases

[0050] According to the invention all Termamy-like alpha-amylases, asdefined above, may be used as the parent (i.e., backbone) alpha-amylase.In a preferred embodiment of the invention the parent alpha-amylase isderived from B. licheniformis, e.g., one of those referred to above,such as the B. licheniformis alpha-amylase having the amino acidsequence shown in SEQ ID NO: 8.

[0051] Parent Hybrid Termamyl-like Alpha-amylases

[0052] The parent alpha-amylase (i.e., backbone alpha-amylase) may alsobe a hybrid alpha-amylase, i.e., an alpha-amylase, which comprises acombination of partial amino acid sequences derived from at least twoalpha-amylases.

[0053] The parent hybrid alpha-amylase may be one, which on the basis ofamino acid homology (identity) and/or DNA hybridization (as definedabove) can be determined to belong to the Termamyl-like alpha-amylasefamily. In this case, the hybrid alpha-amylase is typically composed ofat least one part of a Termamyl-like alpha-amylase and part(s) of one ormore other alpha-amylases selected from Termamyl-like alpha-amylases ornon-Termamyl-like alpha-amylases of microbial (bacterial or fungal)and/or mammalian origin.

[0054] Thus, the parent hybrid alpha-amylase may comprise a combinationof partial amino acid sequences deriving from at least two Termamyl-likealpha-amylases, or from at least one Termamyl-like and at least onenon-Termamyl-like bacterial alpha-amylase, or from at least oneTermamyl-like and at least one fungal alpha-amylase. The Termamyl-likealpha-amylase from which a partial amino acid sequence derives, may beany of the specific Termamyl-like alpha-amylase referred to herein.

[0055] For instance, the parent alpha-amylase may comprise a C-terminalpart of an alpha-amylase derived from a strain of B. licheniformis, anda N-terminal part of an alpha-amylase derived from a strain of B.amyloliquefaciens or from a strain of B. stearothermophilus. Forinstance, the parent alpha-amylase may comprise at least 430 amino acidresidues of the C-terminal part of the B. licheniformis alpha-amylase,and may, e.g., comprise a) an amino acid segment corresponding to the 37N-terminal amino acid residues of the B. amyloliquefaciens alpha-amylasehaving the amino acid sequence shown in SEQ ID NO: 10 and an amino acidsegment corresponding to the 445 C-terminal amino acid residues of theB. licheniformis alpha-amylase having the amino acid sequence shown inSEQ ID NO: 8, or a hybrid Termamyl-like alpha-amylase being identical tothe Termamyl sequence, i.e., the Bacillus licheniformis alpha-amylaseshown in SEQ ID NO: 8, except that the N-terminal 35 amino acid residues(of the mature protein) has been replaced by the N-terminal 33 residuesof BAN (mature protein), i.e., the Bacillus amyloliquefaciensalpha-amylase shown in SEQ ID NO: 10; or b) an amino acid segmentcorresponding to the 68 N-terminal amino acid residues of the B.stearothermophilus alpha-amylase having the amino acid sequence shown inSEQ ID NO: 6 and an amino acid segment corresponding to the 415C-terminal amino acid residues of the B. licheniformis alpha-amylasehaving the amino acid sequence shown in SEQ ID NO: 8.

[0056] Another suitable parent hybrid alpha-amylase is the onepreviously described in WO 96/23874 (from Novo Nordisk) constituting theN-terminus of BAN, Bacillus amyloliquefaciens alpha-amylase (amino acids1-300 of the mature protein) and the C-terminus from Termamyl (aminoacids 301-483 of the mature protein).

[0057] In a preferred embodiment of the invention the parentTermamyl-like alpha-amylase is a hybrid alpha-amylase of SEQ ID NO: 8and SEQ ID NO: 10. Specifically, the parent hybrid Termamyl-likealpha-amylase may be a hybrid alpha-amylase comprising the 445C-terminal amino acid residues of the B. licheniformis alpha-amylaseshown in SEQ ID NO: 8 and the 37 N-terminal amino acid residues of thealpha-amylase derived from B. amyloliquefaciens shown in SEQ ID NO: 10,which may suitably further have the following mutations:H156Y+A181T+N190F+A209V+Q264S (using the numbering in SEQ ID NO: 8). Thelatter mentioned hybrid is used in the examples below and is referred toas LE174.

[0058] Other specifically contemplated parent alpha-amylase includeLE174 with fewer mutations, i.e., the right above mentioned hydridhaving the following mutations: A181T+N190F+A209V+Q264S;N190F+A209V+Q264S; A209V+Q264S; Q264S; H156Y+N190F+A209V+Q264S;H156Y+A209V+Q264S; H156Y+Q264S; H156Y+A181T+A209V+Q264S;H156Y+A181T+Q264S; H156Y+Q264S; H156Y+A181T+N190F+Q264S;H156Y+A181T+N190F; H156Y+A181T+N190F+A209V. These hybrids are alsoconsidered to be part of the invention.

[0059] In a preferred embodiment the parent Termamyl-like alpha amylaseis LE174, SP722, or AA560 including any of

[0060] LE174+G48A+T49I+G107A+I201F; LE174+M197L;LE174+G48A+T49I+G107A+M197L+I201F, or SP722+D183*+G184*;SP722+D183*+G184*+N195F; SP722+D183*+G184*+M202L;SP722+D183*+G184*+N195F+M202L; BSG+I181*+G182*; BSG+I181*+G182*+N193F;BSG+I181*+G182*+M200L; BSG+I181*+G182*+N193F+M200L; AA560+D183*+G184*;AA560+D183*+G184*+N195F; AA560+D183*+G184*+M202L;AA560+D183*+G184*+N195F+M202L.

[0061] Other parent alpha-amylases contemplated include LE429, which isLE174 with an additional substitution in I201F. According to theinvention LE335 is the alpha-amylase, which in comparison to LE429 hasadditional substitutions in T49I+G107A; LE399 is LE335+G48A, i.e.,LE174, with G48A+T49I+G107A+I201F.

[0062] Altered Properties

[0063] The following section discusses the relationship betweenmutations, which are present in variants of the invention, and desirablealterations in properties (relative to those of a parent Termamyl-likealpha-amylase), which may result therefrom.

[0064] As mentioned above the invention relates to Termamyl-likealpha-amylases with altered properties (as mentioned above), inparticular at high temperatures and/or at low pH, in particular at lowcalcium concentrations.

[0065] In the context of the present invention “high temperature” meanstemperatures from 70-120° C., preferably 80-100° C., especially 85-95°C.

[0066] In the context of the present invention the term “low pH” meansfrom a pH in the range from 4-6, preferably 4.2-5.5, especially 4.5-5.

[0067] In the context of the present invention the term “high pH” meansfrom a pH in the range from 8-11, especially 8.5-10.6.

[0068] In the context of the present invention the term “low calciumconcentration” means free calcium levels lower than 60 ppm, preferably40 ppm, more preferably 25 ppm, especially 5 ppm calcium.

[0069] Parent Termamyl-like alpha-amylase specifically contemplated inconnection with going through the specifically contemplated alteredproperties are the above mentioned parent Termamyl-like alpha-amylaseand parent hydrid Termamyl-like alpha-amylases.

[0070] The Termamyl® alpha-amylase is used as the starting point, butcorresponding positions in, e.g., the SP722, BSG, BAN, AA560, SP690, KSMAP1378, and #707 should be understood as disclosed and specificallycomtemplated too.

[0071] In a preferred embodiment the variant of the invention has inparticular at high temperatures and/or at low pH.

[0072] In an aspect the invention relates to variant with alteredproperties as mentioned above.

[0073] In the first aspect a variant of a parent Termamyl-likealpha-amylase, comprising an alteration at one or more positions (usingSEQ ID NO: 8 for the amino acid numbering) selected from the group of:

[0074] 49, 60, 104, 132, 161, 170, 176, 179, 180, 181, 183, 200, 203,204, 207, 212, 237, 239, 250, 280, 298, 318, 374, 385, 393, 402, 406,427, 430, 440, 444, 447, 482,

[0075] wherein

[0076] (a) the alteration(s) are independently

[0077] (i) an insertion of an amino acid downstream of the amino acidwhich occupies the position,

[0078] (ii) a deletion of the amino acid which occupies the position, or

[0079] (iii) a substitution of the amino acid which occupies theposition with a different amino acid,

[0080] (b) the variant has alpha-amylase activity and (c) each positioncorresponds to a position of the amino acid sequence of the parentTermamyl-like alpha-amylase having the amino acid sequence shown in SEQID NO: 8.

[0081] In Termamyl® (SEQ ID NO: 8) such corresponding positions are:

[0082] T49; D60; N104; E132; D161; K170; K176; G179; K180; A181; D183;D200; Y203; D204; D207; I212; K237; S239; E250; N280; Q298; L318; Q374;E385; Q393; Y402; H406; L427 D430; V440; N444; E447; Q482.

[0083] In SP722 (SEQ ID NO: 4) the corresponding positions are:

[0084] T51; D62; N106; D134; D163; Q172; K179; G184; K185; A186; D188;D205; M208; D209; X212; L217, K242, S244, N255, N285, S303, M323; D387,N395; Y404; H408; I429; D432; V442; K446; Q449; K484.

[0085] Corresponding positions in other parent alpha-amylases can befound by alignment as described above and shown in the alignment in FIG.1.

[0086] In a preferred embodiment the variant of the invention (using SEQID NO: 8 (Termamyl™) for the numbering) has one or more of the followingsubstitutions:

[0087] T49I; D60N; N104D; E132A,V,P; D161N; K170Q; K176R; G179N; K180T;A181N; D183N; D200N; X203Y; D204S; D207V,E,L,G; X212I; K237P; S239W;E250G,F; N280S; X298Q; L318M; Q374R; E385V; Q393R; Y402F; H406L,W; L427TD430N; V440A; N444R,K; E447Q,K; Q482K.

[0088] In a preferred embodiment the variant of the invention (using SEQID NO: 4 (SP722) for the numbering) has one or more of the followingsubstitutions:

[0089] T51I; D62N; N106D; D134A,V,P; D163N; X172Q; K179R; G184N; K185T;A186N; D188N; D205N; M208Y; D209S; X212V,E,L,G; L217I, K242P, S244W,N255G,F, N285S, S303Q, X323M; D387V, N395R; Y404F; H408L,W; X429I;D432N; V442A; X446R,K; X449Q,K; X484K, using SEQ ID NO: 4 (SP722) forthe numbering.

[0090] Preferred double, triple and multi-mutations—using SEQ ID NO: 8as the basis for the numbering—are selected from the group consistingof:

[0091] T49I+D60N; T49I+D60N+E132A; T49I+D60N+E132V;T491+D60N+E132V+K170Q; T49I+D60N+E132A+K170Q;T49I+D60N+E132V+K170Q+K176R; T49I+D60N+E132A+K170Q+K176R;T49I+D60N+El32V+K170Q+K176R+D207V; T49I+D60N+E132A+K170Q+K176R+D207V;T49I+D60N+E132V+K170Q+K176R+D207E; T49I+D60N+E132A+K170Q+K176R+D207E;T49I+D60N+E132V+K170OQ+K176R+D207V+E250G;T49I+D60N+E132A+K170Q+K176R+D207V+E250G;T49I+D60N+E132V+K170Q+K176R+D207E+E250G;T49I+D60N+E132A+K170Q+K176R+D207E+E250G;T49I+D60N+E132V+K170Q+K176R+D207E+E250G+N280S;T49I+D60N+E132A+K170Q+K176R+D207E+E250G+N280S;T49I+D60N+E132V+K170Q+K176R+D207V+E250G+N280S;T49I+D60N+E132A+K170Q+K176R+D207V+E250G+N280S;T49I+D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M;T49I+D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M;T49I+D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M;T49I+D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M;T49I+D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R;T49I+D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R;T49I+D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R;T49I+D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R;T49I+D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V;T49I+D6ON+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V;T49I+D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V;T49I+D60N+E132A+K170Q+K176R+D207E+E250G+N28OS+L318M+Q374R+E385V;T49I+D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R;T49I+D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R;T49I+D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R;T49I+D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+Q393R;T49I+D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q373R+E385V+Q393R+Y402F;T49I+D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F;T49I+D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F;T49I+D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+385V+Q393R+Y402F;T49I+D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q373R+E385V+Q393R+Y402F+H406L;T49I+D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L;T49I+D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L;T49I+D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L;T49I+D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q373R+E385V+Q393R+Y402F+H406L+L427I;T49I+D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I;T49I+D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I;T49I+D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E38V+Q393R+Y402F+H406L+L427I;T49I+D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q373R+E385V+Q393R+Y402F+H406L+L427I+V440A;T49I+D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A;T49I+D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A;T49I+D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A;D60N+E132A; D60N+E132V; D60N+E132V+K170Q; D60N+E132A+K170Q;D60N+E132V+K170Q+K176R; T49I+D60N+E132A+K170Q+K176R;D60N+E132V+K170Q+K176R+D207V; T49I+D60N+E132A+K170Q+K176R+D207V;D60N+E132V+K170Q+K176R+D207E; T49I+D60N+E132A+K170Q+K176R+D207E;D60N+E132V+K170Q+K176R+D207V+E250G; D60N+E132A+K170Q+K176R+D207V+E250G;D60N+E132V+K170Q+K176R+D207E+E250G; D60N+E132A+K170Q+K176R+D207E+E250G;D60N+E132V+K170Q+K176R+D207V+E250G+N280S;D60N+E132A+K170Q+K176R+D207V+E250G+N280S;D60N+E132V+K170Q+K176R+D207E+E250G+N280S;D60N+E132A+K170Q+K176R+D207E+E250G+N280S;D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M;D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M;D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M;D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M;D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R;D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R;D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R;D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R;D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V;D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V;D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V;D60N+E132A+K170Q+K17 6R+D207E+E250G+N280S+L318M+Q374R+E385V;D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q373R+E385V+Q393R+Y402F;D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F;D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F;D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F;D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q373R+E385V+Q393R+Y402F+H406L;D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L;D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L;D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L;D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q373R+E385V+Q393R+Y402F+H406L+L427I;D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I;D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I;D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385R+Q393R+Y402F+H406L+L427I;D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q373R+E385V+Q393R+Y402F+H406L+L427I+V440A;D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A;D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A;D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A;E132V+K170Q; E132A+K170Q; E132V+K170Q+K176R; E132A+K170Q+K176R;E132V+K170Q+K176R+D207V; E132A+K170Q+K176R+D207V;E132V+K170Q+K176R+D207E; E132A+K170Q+K176R+D207E;E132V+K170Q+K176R+D207V+E250G; E132A+K170Q+K176R+D207V+E250G;E132V+K170Q+K176R+D207E+E250G; E132A+K170Q+K176R+D207E+E250G;E132V+K170Q+K176R+D207E+E250G+N280S;E132A+K170Q+K176R+D207E+E250G+N280S;E132V+K170Q+K176R+D207V+E250G+N280S;E132A+K170Q+K176R+D207V+E250G+N280S;E132V+K170Q+K176R+D207V+E250G+N280S+L318M;E132A+K170Q+K176R+D207V+E250G+N280S+L318M;E132V+K170Q+K176R+D207E+E250G+N280S+L318M;E132A+K170Q+K176R+D207E+E250G+N280S+L318M;E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R;E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R;E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R;E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R;E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V;E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V;E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V;E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V;E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q373R+E385V+Q393R;E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R;E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R;E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q373R;E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q373R+E385V+Q393R+Y402F;E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F;E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F;E132A+K170Q+K176R+D207E+E250G+N28DS+L318M+Q374R+E385V+Q393R+Y402F;E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q373R+E385V+Q393R+Y402F+H406L;E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L;E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L;E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L;E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q373R+E385V+Q393R+Y402F+H406L+L427I;E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I;E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I;E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I;E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q373R+E385V+Q393R+Y402F+H406L+L427I+V440A;E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A;E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A;E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A;K170Q+K176R; K170Q+K176R+D207V; K170Q+K176R+D207E;K170Q+K176R+D207V+E250G; K170Q+K176R+D207E+E250G;K170Q+K176R+D207V+E250G+N280S; K170Q+K176R+D207E+E250G+N280S;K170Q+K176R+D207E+E250G+N280S+L318M;K170Q+K176R+D207V+E250G+N280S+L318M;K170Q+K176R+D207E+E250G+N280S+L318M+Q374R;K170Q+K176R+D207V+E250G+N280S+L318M+Q374R;K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V;K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V;K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R;K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R;K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F;K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F;K170Q+K176R+D207V+E250G+N280S+L318M+Q373R+385V+Q393R+Y402F+H406L;K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L;K170Q+K176R+D207V+E250G+N280S+L318M+Q373R+E385V+Q393R+Y402F+H406L+L427I;K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I;K170Q+K176R+D207V+E250G+N280S+L318M+Q373R+E385V+Q393R+Y402F+H406L+L427I+V440A;K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A;K176R+D207V; K176R+D207E; K176R+D207V+E250G;K176R+D207E+E250G;K176R+D207V+E250G+N280S; K176R+D207E+E250G+N280S;K176R+D207E+E250G+N280S+L318M; K176R+D207V+E250G+N280S+L318M;K176R+D207E+E250G+N280S+L318M+Q374R;K176R+D207V+E250G+N280S+L318M+Q374R;K176R+D207E+E250G+N280S+L318M+Q374R+E385V;K176R+D207V+E250G+N280S+L318M+Q374R+E385V;K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R;K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R;K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F;K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F;K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L;K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I;K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I;K176R+D207V+E250G+N280S+L318M+Q373R+E385V+Q393R+Y402F+H406L+L427I+V440A;K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A;D207V+E250G; D207E+E250G; D207V+E250G+N280S; D207E+E250G+N280S+L318M;D207V+E250G+N280S+L318M;D207E+E250G+N280S+L318M+Q374R;D207V+E250G+N280S+L318M+Q374R; D207E+E250G+N280S+L318M+Q374R+E385V;D207V+E250G+N280S+L318M+Q374R+E385V;D207V+E250G+N280S+L318M+Q374R+E385V+Q393R;D207E+E250G+N280S+L318M+Q374R+E385V+Q393R;D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F;D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F;D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L;D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L;D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I;D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I;D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A;D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A;E250G+N280S; E250G+N280S+L318M; E250G+N280S+L318M+Q374R;E250G+N280S+L318M+Q374R+E385V; E250G+N280S+L318M+Q374R+E385V+Q393R;E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F;E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L;E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I;E250G+N280S+L318M+Q373R+E385V+Q393R+Y402F+H406L+L427I+V440A;N280S+L318M; N280S+L318M+Q374R; N280S+L318M+Q374R+E385V;N280S+L318M+Q374R+E385V+Q393R; N280S+L318M+Q374R+E385V+Q393R+Y402F;N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L;N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I;N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A; L318M+Q374R;L318M+Q374R+E385V; L318M+Q374R+E385V+Q393R;L318M+Q374R+E385V+Q393R+Y402F; L318M+Q374R+E385V+Q393R+Y402F+H406L;L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I;L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A; Q374R+E385V;Q374R+E385V+Q393R; Q374R+E385V+Q393R+Y402F;Q374R+E385V+Q393R+Y402F+H406L; Q374R+E385V+Q393R+Y402F+H406L+L427I;Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A; E385V+Q393R;E385V+Q393R+Y402F; E385V+Q393R+Y402F+H406L;E385V+Q393R+Y402F+H406L+L427I; E385V+Q393R+Y402F+H406L+L427I+V440A;Q393R+Y402F; Q393R+Y402F+H406L; Q393R+Y402F+H406L+L427I;Q393R+Y402F+H406L+L427I+V440A; Y402F+H406L; Y402F+H406L+L427I;Y402F+H406L+L427I+V440A; H406L+L427I; H406L+L427I+V440A; L427I+V440A;N104D+D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+N444K+E447Q+Q482K;D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+N444K+E447Q+Q482K;D161N+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+N444K+E447Q+Q482K;D161N+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+E447Q+Q482K;N104D+D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+E447Q+Q482K;D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+E447Q+Q482K;N104D+D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N;D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N;H406W+D430N; N444K+E447Q+Q482K; E447Q+Q482K;N104D+D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+N444R+N444K+E447K+Q482K;D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+N444R+N444K+E447K+Q482K;N104D+D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W;D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W; H406W+D430N;N444K+E447K+Q482K; E447K+Q482K;N104D+D161N+A181N+D183N+D200N+D204S+K237P+S239W;N104D+D161N+A181N+D183N+D200N+D204S+K237P;N104D+D161N+A181N+D183N+D200N+D204S;D161N+A181N+D183N+D200N+D204S+K237P+S239W;D161N+A181N+D183N+D200N+D204S+K237P; D161N+A181N+D183N+D200N+D204S;K237P+S239W, using SEQ ID NO: 8 for the numbering.

[0092] In a preferred embodiment the variant has the followingsubstitutions: K170Q+D207V+N280S; E132A+D207V; D207E+E250G+H406L+L427I;D207V+L318M; D60N+D207V+L318M; T49I+E132V+V440A;T491I+K176R+D207V+Y402F; Q374R+E385V+Q393R; N190F+A209V+Q264S;G48A+T491I+G107A+I201F; T491I+G107A+I201F; G48A+T49I+I201F;G48A+T491I+G107A; T49I+I201F; T49I+G107A; G48A+T49I;D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+N444K+E447Q+Q482Kusing SEQ ID NO: 8 for the numbering. Specific variant include: LE399;LE174+G48A+T49I+G107A; LE174+G48A+T49I+I201F; LE174+G48A+G107A+I201F;LE174+T49I+G107A+I201F; LE174+G48A+T49I; LE174+G48A; LE174+G107A+I201F;LE174+I201F, are specifically contemplated variants of the invention.

[0093] Stability

[0094] In the context of the present invention, mutations (includingamino acid substitutionsa and deletion) of importance with respect toachieving altered stability, in particular improved stability (i.e.,higher or lower), at especially high temperatures (i.e., 70-120° C.)and/or extreme pH (i.e. low or high pH, i.e, pH 4-6 or pH 8-11,respectively), in particular at free (i.e., unbound, therefore insolution) calcium concentrations below 60 ppm, include any of themutations listed in the “Altered properties” section. The stability maybe determined as described in the “Materials & Methods” section below.

[0095] General Mutations in Variants of the Invention

[0096] A variant of the invention may in one embodiment comprise one ormore modifications in addition to those outlined above. Thus, it may beadvantageous that one or more Proline (Pro) residues present in the partof the alpha-amylase variant which is modified is/are replaced with anon-Proline residue which may be any of the possible, naturallyoccurring non-Proline residues, and which preferably is an Alanine,Glycine, Serine, Threonine, Valine or Leucine.

[0097] Analogously, in one embodiment one or more Cysteine residuespresent in the parent alpha-amylase may be replaced with a non-Cysteineresidue such as Serine, Alanine, Threonine, Glycine, Valine or Leucine.

[0098] Furthermore, a variant of the invention may—either as the onlymodification or in combination with any of the above outlinedmodifications—be modified so that one or more Asp and/or Glu present inan amino acid fragment corresponding to the amino acid fragment 185-209of SEQ ID NO: 10 is replaced by an Asn and/or Gln, respectively. Also ofinterest is the replacement, in the Termamyl-like alpha-amylase, of oneor more of the Lys residues present in an amino acid fragmentcorresponding to the amino acid fragment 185-209 of SEQ ID NO: 10 by anArg.

[0099] It is to be understood that the present invention encompassesvariants incorporating two or more of the above outlined modifications.

[0100] Furthermore, it may be advantageous to introduce mutations in oneor more of the following positions (using SEQ ID NO: 8 (Termamyl) forthe numbering):

[0101] M15, V128, A111, H133, W138, T149, M197, N188, A209, A210, H405,T412, in particular the following single, double or triple or multimutations:

[0102] M15X, in particular M15T,L;

[0103] V128X, in particular V128E;

[0104] H133X, in particular H133Y;

[0105] N188X, in particular N188S,T,P;

[0106] M197X, in particular M197T,L;

[0107] A209X, in particular A209V;

[0108] M197T/W138F; M197T/W138Y; M15T/H133Y/N188S;

[0109] M15/V128E/H133Y/N188S; E119C/S130C; D124C/R127C; H133Y/T149I;

[0110] G475R, H133Y/S187D; H133Y/A209V.

[0111] Methods for Preparing Alpha-amylase Variants of the Invention

[0112] Several methods for introducing mutations into genes are known inthe art. After a brief description of cloning of alpha-amylase-encodingDNA sequences, methods for generating mutations at specific sites withinthe alpha-amylase-encoding sequence will be discribed.

[0113] Cloning a DNA Sequence Encoding an Alpha-amylase

[0114] The DNA sequence encoding a parent alpha-amylase may be isolatedfrom any cell or microorganism producing the alpha-amylase in question,using various methods well known in the art. First, a genomic DNA and/orcDNA library should be constructed using chromosomal DNA or messengerRNA from the organism that produces the alpha-amylase to be studied.Then, if the amino acid sequence of the alpha-amylase is known,homologous, labeled oligonucleotide probes may be synthesized and usedto identify alpha-amylase-encoding clones from a genomic libraryprepared from the organism in question. Alternatively, a labeledoligonucleotide probe containing sequences homologous to a knownalpha-amylase gene could be used as a probe to identifyalpha-amylase-encoding clones, using hybridization and washingconditions of lower stringency.

[0115] Yet another method for identifying alpha-amylase-encoding cloneswould involve inserting fragments of genomic DNA into an expressionvector, such as a plasmid, transforming alpha-amylase-negative bacteriawith the resulting genomic DNA library, and then plating the transformedbacteria onto agar containing a substrate for alpha-amylase, therebyallowing clones expressing the alpha-amylase to be identified.

[0116] Alternatively, the DNA sequence encoding the enzyme may beprepared synthetically by established standard methods, e.g., thephosphoroamidite method described by S. L. Beaucage and M. H. Caruthers,Tetrahedron Letters 22, 1981, pp. 1859-1869, or the method described byMatthes et al., The EMBO J. 3, 1984, pp. 801-805. In thephosphoroamidite method, oligonucleotides are synthesized, e.g., in anautomatic DNA synthesizer, purified, annealed, ligated and cloned inappropriate vectors.

[0117] Finally, the DNA sequence may be of mixed genomic and syntheticorigin, mixed synthetic and cDNA origin or mixed genomic and cDNAorigin, prepared by ligating fragments of synthetic, genomic or cDNAorigin (as appropriate, the fragments corresponding to various parts ofthe entire DNA sequence), in accordance with standard techniques. TheDNA sequence may also be prepared by polymerase chain reaction (PCR)using specific primers, for instance as described in U.S. Pat. No.4,683,202 or R. K. Saiki et al., Science 239, 1988, pp. 487-491.

[0118] Site-directed Mutagenesis

[0119] Once an alpha-amylase-encoding DNA sequence has been isolated,and desirable sites for mutation identified, mutations may be introducedusing synthetic oligonucleotides. These oligonucleotides containnucleotide sequences flanking the desired mutation sites; mutantnucleotides are inserted during oligonucleotide synthesis. In a specificmethod, a single-stranded gap of DNA, bridging thealpha-amylase-encoding sequence, is created in a vector carrying thealpha-amylase gene. Then the synthetic nucleotide, bearing the desiredmutation, is annealed to a homologous portion of the single-strandedDNA. The remaining gap is then filled in with DNA polymerase I (Klenowfragment) and the construct is ligated using T4 ligase. A specificexample of this method is described in Morinaga et al. (1984). U.S. Pat.No. 4,760,025 disclose the introduction of oligonucleotides encodingmultiple mutations by performing minor alterations of the cassette.However, an even greater variety of mutations can be introduced at anyone time by the Morinaga method, because a multitude ofoligonucleotides, of various lengths, can be introduced.

[0120] Another method for introducing mutations intoalpha-amylase-encoding DNA sequences is described in Nelson and Long(1989). It involves the 3-step generation of a PCR fragment containingthe desired mutation introduced by using a chemically synthesized DNAstrand as one of the primers in the PCR reactions. From thePCR-generated fragment, a DNA fragment carrying the mutation may beisolated by cleavage with restriction endonucleases and reinserted intoan expression plasmid.

[0121] Alternative methods for providing variants of the inventioninclude gene shuffling, e.g., as described in WO 95/22625 (from AffymaxTechnologies N.V.) or in WO 96/00343 (from Novo Nordisk A/S), or othercorresponding techniques resulting in a hybrid enzyme comprising themutation(s), e.g., substitution(s) and/or deletion(s), in question.Examples of parent alpha-amylases, which suitably may be used forproviding a hybrid with the desired mutations(s) according to theinvention include the KSM-K36 and KSM-K38 alpha-amylases disclosed in EP1,022,334 (hereby incorporated by reference).

[0122] Expression of Alpha-amylase Variants

[0123] According to the invention, a DNA sequence encoding the variantproduced by methods described above, or by any alternative methods knownin the art, can be expressed, in enzyme form, using an expression vectorwhich typically includes control sequences encoding a promoter,operator, ribosome binding site, translation initiation signal, and,optionally, a repressor gene or various activator genes.

[0124] The recombinant expression vector carrying the DNA sequenceencoding an alpha-amylase variant of the invention may be any vector,which may conveniently be subjected to recombinant DNA procedures, andthe choice of vector will often depend on the host cell into which it isto be introduced. Thus, the vector may be an autonomously replicatingvector, i.e., a vector which exists as an extrachromosomal entity, thereplication of which is independent of chromosomal replication, e.g., aplasmid, a bacteriophage or an extrachromosomal element, minichromosomeor an artificial chromosome. Alternatively, the vector may be one which,when introduced into a host cell, is integrated into the host cellgenome and replicated together with the chromosome(s) into which it hasbeen integrated.

[0125] In the vector, the DNA sequence should be operably connected to asuitable promoter sequence. The promoter may be any DNA sequence, whichshows transcriptional activity in the host cell of choice and may bederived from genes encoding proteins either homologous or heterologousto the host cell. Examples of suitable promoters for directing thetranscription of the DNA sequence encoding an alpha-amylase variant ofthe invention, especially in a bacterial host, are the promoter of thelac operon of E.coli, the Streptomyces coelicolor agarase gene dagApromoters, the promoters of the Bacillus licheniformis alpha-amylasegene (amyL), the promoters of the Bacillus stearothermophilus maltogenicamylase gene (amyM), the promoters of the Bacillus amyloliquefaciensalpha-amylase (amyQ), the promoters of the Bacillus subtilis xylA andxylB genes etc. For transcription in a fungal host, examples of usefulpromoters are those derived from the gene encoding A. oryzae TAKAamylase, Rhizomucor miehei aspartic proteinase, A. niger neutralalpha-amylase, A. niger acid stable alpha-amylase, A. nigerglucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A.oryzae triose phosphate isomerase or A. nidulans acetamidase.

[0126] The expression vector of the invention may also comprise asuitable transcription terminator and, in eukaryotes, polyadenylationsequences operably connected to the DNA sequence encoding thealpha-amylase variant of the invention. Termination and polyadenylationsequences may suitably be derived from the same sources as the promoter.

[0127] The vector may further comprise a DNA sequence enabling thevector to replicate in the host cell in question. Examples of suchsequences are the origins of replication of plasmids pUC19, pACYC177,pUB110, pE194, pAMB1 and pIJ702.

[0128] The vector may also comprise a selectable marker, e.g. a gene theproduct of which complements a defect in the host cell, such as the dalgenes from B. subtilis or B. licheniformis, or one which confersantibiotic resistance such as ampicillin, kanamycin, chloramphenicol ortetracyclin resistance. Furthermore, the vector may comprise Aspergillusselection markers such as amdS, argB, niaD and sC, a marker giving riseto hygromycin resistance, or the selection may be accomplished byco-transformation, e.g., as described in WO 91/17243.

[0129] While intracellular expression may be advantageous in somerespects, e.g., when using certain bacteria as host cells, it isgenerally preferred that the expression is extracellular. In general,the Bacillus alpha-amylases mentioned herein comprise a preregionpermitting secretion of the expressed protease into the culture medium.If desirable, this preregion may be replaced by a different preregion orsignal sequence, conveniently accomplished by substitution of the DNAsequences encoding the respective preregions.

[0130] The procedures used to ligate the DNA construct of the inventionencoding an alpha-amylase variant, the promoter, terminator and otherelements, respectively, and to insert them into suitable vectorscontaining the information necessary for replication, are well known topersons skilled in the art (cf., for instance, Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor,1989).

[0131] The cell of the invention, either comprising a DNA construct oran expression vector of the invention as defined above, isadvantageously used as a host cell in the recombinant production of analpha-amylase variant of the invention. The cell may be transformed withthe DNA construct of the invention encoding the variant, conveniently byintegrating the DNA construct (in one or more copies) in the hostchromosome. This integration is generally considered to be an advantageas the DNA sequence is more likely to be stably maintained in the cell.Integration of the DNA constructs into the host chromosome may beperformed according to conventional methods, e.g., by homologous orheterologous recombination. Alternatively, the cell may be transformedwith an expression vector as described above in connection with thedifferent types of host cells.

[0132] The cell of the invention may be a cell of a higher organism suchas a mammal or an insect, but is preferably a microbial cell, e.g., abacterial or a fungal (including yeast) cell.

[0133] Examples of suitable bacteria are Gram-positive bacteria such asBacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillusbrevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacilluslautus, Bacillus megaterium, Bacillus thuringiensis, or Streptomyceslividans or Streptomyces murinus, or gramnegative bacteria such asE.coli. The transformation of the bacteria may, for instance, beeffected by protoplast transformation or by using competent cells in amanner known per se.

[0134] The yeast organism may favorably be selected from a species ofSaccharomyces or Schizosaccharomyces, e.g. Saccharomyces cerevisiae. Thefilamentous fungus may advantageously belong to a species ofAspergillus, e.g., Aspergillus oryzae or Aspergillus niger. Fungal cellsmay be transformed by a process involving protoplast formation andtransformation of the protoplasts followed by regeneration of the cellwall in a manner known per se. A suitable procedure for transformationof Aspergillus host cells is described in EP 238 023.

[0135] In a yet further aspect, the present invention relates to amethod of producing an alpha-amylase variant of the invention, whichmethod comprises cultivating a host cell as described above underconditions conducive to the production of the variant and recovering thevariant from the cells and/or culture medium.

[0136] The medium used to cultivate the cells may be any conventionalmedium suitable for growing the host cell in question and obtainingexpression of the alpha-amylase variant of the invention. Suitable mediaare available from commercial suppliers or may be prepared according topublished recipes (e.g., as described in catalogues of the American TypeCulture Collection).

[0137] The alpha-amylase variant secreted from the host cells mayconveniently be recovered from the culture medium by well-knownprocedures, including separating the cells from the medium bycentrifugation or filtration, and precipitating proteinaceous componentsof the medium by means of a salt such as ammonium sulphate, followed bythe use of chromatographic procedures such as ion exchangechromatography, affinity chromatography, or the like.

[0138] Industrial Applications

[0139] The alpha-amylase variants of this invention possess valuableproperties allowing for a variety of industrial applications. Inparticular, enzyme variants of the invention are applicable as acomponent in washing, dishwashing, and hard surface cleaning detergentcompositions.

[0140] Variant of the invention with altered properties may be used forstarch processes, in particular starch conversion, especiallyliquefaction of starch (see, e.g., U.S. Pat. No. 3,912,590, EP patentpublications Nos. 252 730 and 63 909, WO 99/19467, and WO 96/28567 allreferences hereby incorporated by reference). Also contemplated arecompositions for starch conversion purposes, which may beside thevariant of the invention also comprise a AMG, pullulanase, and otheralpha-amylases.

[0141] Further, variants of the invention are also particularly usefulin the production of sweeteners and ethanol (see, e.g., U.S. Pat. No.5,231,017 hereby incorporated by reference), such as fuel, drinking andindustrial ethanol, from starch or whole grains.

[0142] A variant of the invention may also be used for textile desizing(see, e.g., WO 95/21247, U.S. Pat. No. 4,643,736, EP 119,920 hereby incorporate by reference).

[0143] Detergent Compositions

[0144] As mentioned above, variants of the invention may suitably beincorporated in detergent compositions. Reference is made, for example,to WO 96/23874 and WO 97/07202 for further details concerning relevantingredients of detergent compositions (such as laundry or dishwashingdetergents), appropriate methods of formulating the variants in suchdetergent compositions, and for examples of relevant types of detergentcompositions.

[0145] Detergent compositions comprising a variant of the invention mayadditionally comprise one or more other enzymes, such as a protease, alipase, a peroxidase, another amylolytic enzyme, glucoamylase,maltogenic amylase, CGTase and/or a cellulase, mannanase (such asMannaway™ from Novozymes, Denmark)), pectinase, pectine lyase, cutinase,laccase, and/or another alpha-amylase.

[0146] Alpha-amylase variants of the invention may be incorporated indetergents at conventionally employed concentrations. It is at presentcontemplated that a variant of the invention may be incorporated in anamount corresponding to 0.00001-10 mg (calculated as pure, active enzymeprotein) of alpha-amylase per liter of wash/dishwash liquor usingconventional dosing levels of detergent.

[0147] Compositions

[0148] The invention also related to composition comprising a variant ofthe invention, and in a preferred embodiment also a B.stearothermophilus alpha-amylase (BSG), in particular a variant thereof.

[0149] In another embodient the composition comprises beside a variantof the invention a glucoamylase, in particular a glucoamylaseoriginating from Aspergillus niger (e.g., the G1 or G2 A. niger AMGdisclosed in Boel et al. (1984), “Glucoamylases G1 and G2 fromAspergillus niger are synthesized from two different but closely relatedmRNAs”, EMBO J. 3 (5), p. 1097-1102, or a variant therefore, inparticular a variant disclosed in WO 00/04136 or WO 01/04273 or theTalaromyces emersonii AMG disclosed in WO 99/28448.

[0150] A specific combination is LE399 and a variant disclosed in WO00/04136 or Wo 01/04273, in particular a variant with oe or more of thefollowing substitutions:N9A,S56A,V59A,S119P,A246T,N313G,E342T,A393R,S394R,Y402F,E408R, inparticular a variant with all mutation.

[0151] In an embodiment the composition of the invention also comprisesa pullulanase, in particular a Bacillus pullulanase.

[0152] Materials and Methods

[0153] Enzymes:

[0154]Bacillus licheniformis alpha-amylase shown in SEQ ID NO: 8 andalso available from Novozymes.

[0155] AA560: SEQ ID NO: 12; disclosed in WO 00/60060; deposited on Jan.25, 1999 at DSMZ and assigned the DSMZ no. 12649. AA560 were depositedby the inventors under the terms of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure at Deutshe Sammmlung von Microorganismenund Zellkulturen GmbH (DSMZ), Mascheroder Weg lb, D-38124 BraunschweigDel.

[0156] LB medium (In 1 liter H20: 10 g bacto-tryptone, 5 g bacto-yeastextract, 10 g NaCl, pH adjusted to 7.0 w. NaOH, autoclaved).

[0157] TY agar plates (In 1 liter H20: 16 g bacto-tryptone, 10 gbacto-yeast extract, 5 g NaCl, pH adjusted to 7.0 w. NaOH, and 15 gbacto-agar is added prior to autoclaving).

[0158] 10% Lugol solution (Iodine/Potassium iodine solution; made by10-fold dil. in H20 of stock: Sigma Cat. no. L 6146).

[0159]Bacillus subtilis SHA273: see WO 95/10603

[0160] Plasmids

[0161] pDN1528 contains the complete gene encoding Termamyl, amyl, theexpression of which is directed by its own promoter. Further, theplasmid contains the origin of replication, ori, from plasmid pUB110 andthe cat gene from plasmid pC194 conferring resistance towardschloramphenicol. pDN1528 is shown in FIG. 9 of WO 96/23874.

[0162] Methods:

[0163] Low pH Filter Assay

[0164]Bacillus libraries are plated on a sandwich of cellulose acetate(OE 67, Schleicher & Schuell, Dassel, Germany)—and nitrocellulosefilters (Protran-Ba 85, Schleicher & Schuell, Dassel, Germany) on TYagar plates with 10 micro g/ml chloramphenicol at 37° C. for at least 21hours. The cellulose acetate layer is located on the TY agar plate.

[0165] Each filter sandwich is specifically marked with a needle afterplating, but before incubation in order to be able to localize positivevariants on the filter, and the nitrocellulose filter with boundvariants is transferred to a container with citrate buffer, pH 4.5 andincubated at 80° C. for 20 minutes (when screening for variants in thewild type backbone) or 85° C. for 60 minutes (when screening forvariants in the LE399 backbone). The cellulose acetate filters withcolonies are stored on the TY-plates at room temperature until use.After incubation, residual activity is detected on assay platescontaining 1% agarose, 0.2% starch in citrate buffer, pH 6.0. The assayplates with nitrocellulose filters are marked the same ay as the filtersandwich and incubated for 2 hours at 50° C. After removal of thefilters the assay plates are stained with 10% Lugol solution. Starchdegrading variants are detected as white spots on dark blue backgroundand then identified on the storage plates. Positive variants arere-screened twice under the same conditions as the first screen.

[0166] Secondary Screening

[0167] Positive transformants after rescreening are picked from thestorage plate and tested in a secondary plate assay. Positivetransformants are grown for 22 hours at 37° C. in 5 mlLB+chloramphenicol. The Bacillus culture of each positive transformantand as a control a clone expressing the corresponding backbone areincubated in citrate buffer, pH 4.5 at 90° C. and samples are taken at0, 10, 20, 30, 40, 60 and 80 minutes. A 3 micro liter sample is spottedon an assay plate. The assay plate is stained with 10% Lugol solution.Improved variants are seen as variants with higher residual activity(detected as halos on the assay plate) than the backbone. The improvedvariants are determined by nucleotide sequencing.

[0168] Stability Assay of Unpurified Variants:

[0169] Bacillus cultures expressing the variants to be analysed aregrown for 21 hours at 37° C. in 10 ml LB+chloramphenicol. 800 microliter culture is mixed with 200 micro 1 citrate buffer, pH 4.5. A numberof 70 micro 1 aliquots corresponding to the number of sample time pointsare made in PCR tubes and incubated at 70° C. (for variants in the wtbackbone) or 90° C. (for variants in LE399) for various time points(typically 5, 10, 15, 20, 25 and 30 minutes) in a PCR machine. The 0 minsample is not incubated at high temperature. Activity in the sample ismeasured by transferring 20 micro 1 to 200 micro 1 of the alpha-amylasePNP-G7 substrate MPR3 ((Boehringer Mannheim Cat. no. 1660730) asdescribed below under “Assays for Alpha-Amylase Activity”. Results areplotted as percentage activity (relative to the 0 time point) versustime, or stated as percentage residual activity after incubation for acertain period of time.

[0170] Fermentation and Purification of Alpha-amylase Variants

[0171] A B. subtilis strain harbouring the relevant expression plasmidis streaked on a LB-agar plate with 10 micro g/ml kanamycin from −80° C.stock, and grown overnight at 37° C. The colonies are transferred to 100ml PS-1 media supplemented with 10 micro g/ml chloamphinicol in a 500 mlshaking flask. Composition of PS-1 medium: Pearl sugar 100 g/l Soy BeanMeal 40 g/l Na2HPO4, 12 H2O 10 g/l PluronicTM PE 6100 0.1 g/l CaCO3 5g/l

[0172] The culture is shaken at 37° C. at 270 rpm for 5 days.

[0173] Cells and cell debris are removed from the fermentation broth bycentrifugation at 4500 rpm in 20-25 minutes. Afterwards the supernatantis filtered to obtain a completely clear solution. The filtrate isconcentrated and washed on a UF-filter (10000 cut off membrane) and thebuffer is changed to 20 mM Acetate pH 5.5. The UF-filtrate is applied ona S-sepharose F.F. and elution is carried out by step elution with 0.2MNaCl in the same buffer. The eluate is dialysed against 10 mM Tris, pH9.0 and applied on a Q-sepharose F.F. and eluted with a linear gradientfrom 0-0.3M NaCl over 6 column volumes. The fractions that contain theactivity (measured by the Phadebas assay) are pooled, pH was adjusted topH 7.5 and remaining color was removed by a treatment with 0.5% W/vol.active coal in 5 minutes.

[0174] Stability Determination of Purified Variants

[0175] All stability trials of purified variants are made using the sameset up. The method is as follows: The enzyme is incubated under therelevant conditions (1-4). Samples are taken at various time points,e.g., after 0, 5, 10, 15 and 30 minutes and diluted 25 times (samedilution for all taken samples) in assay buffer (0.1 M 50 mM Brittonbuffer pH 7.3) and the activity is measured using the Phadebas assay(Pharmacia) under standard conditions pH 7.3, 37° C.

[0176] The activity measured before incubation (0 minutes) is used asreference (100%). The decline in percent is calculated as a function ofthe incubation time. The table shows the residual activity after, e.g.,30 minutes of incubation.

[0177] Specific Activity Determination

[0178] The specific activity is determined using the Phadebas assay(Pharmacia) as activity/mg enzyme. The manufactures instructions arefollowed (see also below under “Assay for α-amylase activity).

[0179] Assays for Alpha-Amylase Activity

[0180] 1. Phadebas Assay

[0181] Alpha-amylase activity is determined by a method employingPhadebas® tablets as substrate. Phadebas tablets (Phadebas® a AmylaseTest, supplied by Pharmacia Diagnostic) contain a cross-linked insolubleblue-colored starch polymer, which has been mixed with bovine serumalbumin and a buffer substance and tabletted.

[0182] For every single measurement one tablet is suspended in a tubecontaining 5 ml 50 mM Britton-Robinson buffer (50 mM acetic acid, 50 mMphosphoric acid, 50 mM boric acid, 0.1 mM CaCl2, pH adjusted to thevalue of interest with NaOH). The test is performed in a water bath atthe temperature of interest. The alpha-amylase to be tested is dilutedin x ml of 50 mM Britton-Robinson buffer. 1 ml of this alpha-amylasesolution is added to the 5 ml 50 mM Britton-Robinson buffer. The starchis hydrolyzed by the alpha-amylase giving soluble blue fragments. Theabsorbance of the resulting blue solution, measuredspectrophotometrically at 620 nm, is a function of the alpha-amylaseactivity.

[0183] It is important that the measured 620 nm absorbance after 10 or15 minutes of incubation (testing time) is in the range of 0.2 to 2.0absorbance units at 620 nm. In this absorbance range there is linearitybetween activity and absorbance (Lambert-Beer law). The dilution of theenzyme must therefore be adjusted to fit this criterion. Under aspecified set of conditions (temp., pH, reaction time, bufferconditions) 1 mg of a given alpha-amylase will hydrolyze a certainamount of substrate and a blue colour will be produced. The colourintensity is measured at 620 nm. The measured absorbance is directlyproportional to the specific activity (activity/mg of pure alpha-amylaseprotein) of the alpha-amylase in question under the given set ofconditions.

[0184] 2. Alternative Method

[0185] Alpha-amylase activity is determined by a method employing thePNP-G7 substrate. PNP-G7 which is a abbreviation forp-nitrophenyl-alpha, D-maltoheptaoside is a blocked oligosaccharidewhich can be cleaved by an endo-amylase. Following the cleavage, thealpha-Glucosidase included in the kit digest the substrate to liberate afree PNP molecule which has a yellow colour and thus can be measured byvisible spectophometry at λ=405 nm (400-420 nm). Kits containing PNP-G7substrate and alpha-Glucosidase is manufactured by Boehringer-Mannheim(cat. No.1054635).

[0186] To prepare the reagent solution 10 ml of substrate/buffersolution is added to 50 ml enzyme/buffer solution as recommended by themanufacturer. The assay is performed by transferring 20 micro 1 sampleto a 96 well microtitre plate and incubating at 25° C. 200 micro 1reagent solution pre-equilibrated to 25° C. is added. The solution ismixed and pre-incubated 1 minute and absorption is measured every 30sec. over 4 minutes at OD 405 nm in an ELISA reader.

[0187] The slope of the time dependent absorption-curve is directlyproportional to the activity of the alpha-amylase in question under thegiven set of conditions.

EXAMPLES Example 1

[0188] Construction, by error-prone PCR mutagenesis, of Bacilluslicheniformis alpha-amylase variants having an improved stability at lowpH, high temperature and low calcium ion concentration compared to theparent enzyme.

[0189] Error-prone PCR Mutagenesis and Library Construction

[0190] To improve the stability at low pH and low calcium concentrationof the parent Bacillus licheniformis alpha-amylase, error-prone PCRmutagenesis was performed. The plasmid pDN1528 encoding the wild-typeBacillus licheniformis alpha-amylase gene was utilized as template toamplify this gene with primers: 22149: 5′-CGA TTG CTG ACG CTG TTA TTTGCG-3′ (SEQID NO: 14) and 24814: 5′-GAT CAC CCG CGA TAC CGT C-3′ (SEQ IDNO: 15) under PCR conditions where increased error rates leads tointroduction of random point mutations. The PCR conditions utilizedwere: 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 4 mM MgCl2, 0.3 mM MnCl2, 0.1mM dGTP/dATP, 0.5 mM dTTP/dCTP, and 2.5 units Taq polymerase per 100micro 1 reaction.

[0191] The resultant PCR fragment was purified on gel and used in aPCR-based multimerization step with a gel purified vector fragmentcreated by PCR amplification of pDN1528 with primers #24: 5′-GAA TGT ATGTCG GCC GGC AAA ACG CCG GTG A-3′ (SEQ ID NO: 16) and #27: 5′-GCC GCC GCTGCT GCA GAA TGA GGC AGC AAG-3′ (SEQ ID NO:17) forming an overlap to theinsert fragment. The multimerization reaction was subsequentlyintroduced into B. subtilis (Shafikhani et al., Biotechniques, 23(1997), 304-310).

[0192] Screening

[0193] The error-prone library described above was screened in the lowpH filter assay (see “Materials & Methods”). Clones testing positiveupon rescreening was submitted to secondary screening for stability inthe liquid assay described in Materials and Methods.

[0194] Results:

[0195] Increased stability at pH 4.5, 5 ppm calcium incubated at 90° C.Name wt LE488 LE489 7.19.1 8.9.1 Mutations − D207V K170Q E132A D207ED207V D207V E250G N280S H406L L427I Stability¹⁾ − + + + +

[0196] Increased stability at pH 4.5, 5 ppm calcium incubated at 90° C.Name wt LE491 LE492 LE493 LE494 19.3.1 Mutations — D60N T49I T49I Q374RN190F D207V E132V K176R E385V A209V L318M V440A D207V Q393R Q264S Y402FStabi- — + + + + + lity 1)

[0197] Increased stability at pH 4.5, 5 ppm calcium incubated at 90° C.Name wt E132-1 D207-7 D207-6 E250-8 Mutations − E132P D207L D207G E250FStability¹⁾ − + + + +

Example 2

[0198] Transfer, by site-directed mutagenesis, of a selection ofmutations from Example 1 to a new (non-wild type) backbone to improvestability at low pH and low calcium ion concentration compared to theparent enzyme.

[0199] Site-directed Mutagenesis

[0200] Mutations from LE493 (K176R+D207V+Y402F) were transferred toLE399 yielding LE495. This was performed by the overlap PCR method(Kirchhoff and Desrosiers, PCR Methods and Applications, 2 (1993),301-304). 2 overlapping PCR fragments were generated by amplification ofthe LE399 template with the primers: Fragment A: #312 Mut176 5′-CCC GAAAGC TGA ACC GCA TCT ATA GGT TTC AAG GGA AGA CTT GGG ATT-3′ (SEQ ID NO:18) (mutated codon indicated in bold) and #290 D207overlap 5′-AGG ATGGTC ATA ATC AA GTC GG-3′ (SEQ ID NO: 19); Fragment B: #313 Mut207 5′-CCGACT TTG ATT ATG ACC ATC CTG TTG TCG TAG CAG AGA TTA AGA GAT GGG G-3′(SEQ ID NO: 20) and #314 Mut402 5′-CGA CAA TGT CAT GGT GGT CGA AAA AATCAT GCT GTG CTC CGT ACG-3′ (SEQ ID NO: 21). Fragments A and B were mixedin equimolar ratios and subsequently the full-length fragment wasamplified with the external primers: #312 Mut176 and #314 Mut402. Thisfragment was used in a multimerization reaction with the vector PCRfragment created with the primers #296 Y402 multi 5′-TTT CGA CCA CCA TGACAT TGT CG-3′ (SEQ ID NO: 22) and #305 399Multi176 5′-TAT AGA TGC GGTTCA GCT TTC GGG-3′ (SEQ ID NO: 23) on template LE399 as described above.The multimerization reaction was subsequently transformed into B.subtilis. Clones were screened for stability in the assay mentionedabove. The presence of the mutations from LE493 in several clones withincreased stability was confirmed by sequencing.

[0201] LE 497 was obtained in a similar manner by amplifying the LE399encoding template with primers #312 Mut176 and #314 Mut402 and using theresulting PCR fragment in a multimerization reaction with a vectorfragment obtained by PCR amplification of the LE399 template with theprimers #296 Y402multi and #305 399Multi176.

[0202] Results:

[0203] Stabilization of LE399 variant at pH 4.5, 5 ppm calcium incubatedat 9° C. Name LE399 LE495 LE497 Mutations − K176R K176R (backbone) D207VY402F Y402F Stability¹⁾ − + +

[0204]

1 30 1 1455 DNA Bacillus sp. CDS (1)..(1455) 1 cat cat aat gga aca aatggt act atg atg caa tat ttc gaa tgg tat 48 His His Asn Gly Thr Asn GlyThr Met Met Gln Tyr Phe Glu Trp Tyr 1 5 10 15 ttg cca aat gac ggg aatcat tgg aac agg ttg agg gat gac gca gct 96 Leu Pro Asn Asp Gly Asn HisTrp Asn Arg Leu Arg Asp Asp Ala Ala 20 25 30 aac tta aag agt aaa ggg ataaca gct gta tgg atc cca cct gca tgg 144 Asn Leu Lys Ser Lys Gly Ile ThrAla Val Trp Ile Pro Pro Ala Trp 35 40 45 aag ggg act tcc cag aat gat gtaggt tat gga gcc tat gat tta tat 192 Lys Gly Thr Ser Gln Asn Asp Val GlyTyr Gly Ala Tyr Asp Leu Tyr 50 55 60 gat ctt gga gag ttt aac cag aag gggacg gtt cgt aca aaa tat gga 240 Asp Leu Gly Glu Phe Asn Gln Lys Gly ThrVal Arg Thr Lys Tyr Gly 65 70 75 80 aca cgc aac cag cta cag gct gcg gtgacc tct tta aaa aat aac ggc 288 Thr Arg Asn Gln Leu Gln Ala Ala Val ThrSer Leu Lys Asn Asn Gly 85 90 95 att cag gta tat ggt gat gtc gtc atg aatcat aaa ggt gga gca gat 336 Ile Gln Val Tyr Gly Asp Val Val Met Asn HisLys Gly Gly Ala Asp 100 105 110 ggt acg gaa att gta aat gcg gta gaa gtgaat cgg agc aac cga aac 384 Gly Thr Glu Ile Val Asn Ala Val Glu Val AsnArg Ser Asn Arg Asn 115 120 125 cag gaa acc tca gga gag tat gca ata gaagcg tgg aca aag ttt gat 432 Gln Glu Thr Ser Gly Glu Tyr Ala Ile Glu AlaTrp Thr Lys Phe Asp 130 135 140 ttt cct gga aga gga aat aac cat tcc agcttt aag tgg cgc tgg tat 480 Phe Pro Gly Arg Gly Asn Asn His Ser Ser PheLys Trp Arg Trp Tyr 145 150 155 160 cat ttt gat ggg aca gat tgg gat cagtca cgc cag ctt caa aac aaa 528 His Phe Asp Gly Thr Asp Trp Asp Gln SerArg Gln Leu Gln Asn Lys 165 170 175 ata tat aaa ttc agg gga aca ggc aaggcc tgg gac tgg gaa gtc gat 576 Ile Tyr Lys Phe Arg Gly Thr Gly Lys AlaTrp Asp Trp Glu Val Asp 180 185 190 aca gag aat ggc aac tat gac tat cttatg tat gca gac gtg gat atg 624 Thr Glu Asn Gly Asn Tyr Asp Tyr Leu MetTyr Ala Asp Val Asp Met 195 200 205 gat cac cca gaa gta ata cat gaa cttaga aac tgg gga gtg tgg tat 672 Asp His Pro Glu Val Ile His Glu Leu ArgAsn Trp Gly Val Trp Tyr 210 215 220 acg aat aca ctg aac ctt gat gga tttaga ata gat gca gtg aaa cat 720 Thr Asn Thr Leu Asn Leu Asp Gly Phe ArgIle Asp Ala Val Lys His 225 230 235 240 ata aaa tat agc ttt acg aga gattgg ctt aca cat gtg cgt aac acc 768 Ile Lys Tyr Ser Phe Thr Arg Asp TrpLeu Thr His Val Arg Asn Thr 245 250 255 aca ggt aaa cca atg ttt gca gtggct gag ttt tgg aaa aat gac ctt 816 Thr Gly Lys Pro Met Phe Ala Val AlaGlu Phe Trp Lys Asn Asp Leu 260 265 270 ggt gca att gaa aac tat ttg aataaa aca agt tgg aat cac tcg gtg 864 Gly Ala Ile Glu Asn Tyr Leu Asn LysThr Ser Trp Asn His Ser Val 275 280 285 ttt gat gtt cct ctc cac tat aatttg tac aat gca tct aat agc ggt 912 Phe Asp Val Pro Leu His Tyr Asn LeuTyr Asn Ala Ser Asn Ser Gly 290 295 300 ggt tat tat gat atg aga aat atttta aat ggt tct gtg gtg caa aaa 960 Gly Tyr Tyr Asp Met Arg Asn Ile LeuAsn Gly Ser Val Val Gln Lys 305 310 315 320 cat cca aca cat gcc gtt actttt gtt gat aac cat gat tct cag ccc 1008 His Pro Thr His Ala Val Thr PheVal Asp Asn His Asp Ser Gln Pro 325 330 335 ggg gaa gca ttg gaa tcc tttgtt caa caa tgg ttt aaa cca ctt gca 1056 Gly Glu Ala Leu Glu Ser Phe ValGln Gln Trp Phe Lys Pro Leu Ala 340 345 350 tat gca ttg gtt ctg aca agggaa caa ggt tat cct tcc gta ttt tat 1104 Tyr Ala Leu Val Leu Thr Arg GluGln Gly Tyr Pro Ser Val Phe Tyr 355 360 365 ggg gat tac tac ggt atc ccaacc cat ggt gtt ccg gct atg aaa tct 1152 Gly Asp Tyr Tyr Gly Ile Pro ThrHis Gly Val Pro Ala Met Lys Ser 370 375 380 aaa ata gac cct ctt ctg caggca cgt caa act ttt gcc tat ggt acg 1200 Lys Ile Asp Pro Leu Leu Gln AlaArg Gln Thr Phe Ala Tyr Gly Thr 385 390 395 400 cag cat gat tac ttt gatcat cat gat att atc ggt tgg aca aga gag 1248 Gln His Asp Tyr Phe Asp HisHis Asp Ile Ile Gly Trp Thr Arg Glu 405 410 415 gga aat agc tcc cat ccaaat tca ggc ctt gcc acc att atg tca gat 1296 Gly Asn Ser Ser His Pro AsnSer Gly Leu Ala Thr Ile Met Ser Asp 420 425 430 ggt cca ggt ggt aac aaatgg atg tat gtg ggg aaa aat aaa gcg gga 1344 Gly Pro Gly Gly Asn Lys TrpMet Tyr Val Gly Lys Asn Lys Ala Gly 435 440 445 caa gtt tgg aga gat attacc gga aat agg aca ggc acc gtc aca att 1392 Gln Val Trp Arg Asp Ile ThrGly Asn Arg Thr Gly Thr Val Thr Ile 450 455 460 aat gca gac gga tgg ggtaat ttc tct gtt aat gga ggg tcc gtt tcg 1440 Asn Ala Asp Gly Trp Gly AsnPhe Ser Val Asn Gly Gly Ser Val Ser 465 470 475 480 gtt tgg gtg aag caa1455 Val Trp Val Lys Gln 485 2 485 PRT Bacillus sp. 2 His His Asn GlyThr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr 1 5 10 15 Leu Pro AsnAsp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ala 20 25 30 Asn Leu LysSer Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp 35 40 45 Lys Gly ThrSer Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60 Asp Leu GlyGlu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly 65 70 75 80 Thr ArgAsn Gln Leu Gln Ala Ala Val Thr Ser Leu Lys Asn Asn Gly 85 90 95 Ile GlnVal Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110 GlyThr Glu Ile Val Asn Ala Val Glu Val Asn Arg Ser Asn Arg Asn 115 120 125Gln Glu Thr Ser Gly Glu Tyr Ala Ile Glu Ala Trp Thr Lys Phe Asp 130 135140 Phe Pro Gly Arg Gly Asn Asn His Ser Ser Phe Lys Trp Arg Trp Tyr 145150 155 160 His Phe Asp Gly Thr Asp Trp Asp Gln Ser Arg Gln Leu Gln AsnLys 165 170 175 Ile Tyr Lys Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp GluVal Asp 180 185 190 Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala AspVal Asp Met 195 200 205 Asp His Pro Glu Val Ile His Glu Leu Arg Asn TrpGly Val Trp Tyr 210 215 220 Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg IleAsp Ala Val Lys His 225 230 235 240 Ile Lys Tyr Ser Phe Thr Arg Asp TrpLeu Thr His Val Arg Asn Thr 245 250 255 Thr Gly Lys Pro Met Phe Ala ValAla Glu Phe Trp Lys Asn Asp Leu 260 265 270 Gly Ala Ile Glu Asn Tyr LeuAsn Lys Thr Ser Trp Asn His Ser Val 275 280 285 Phe Asp Val Pro Leu HisTyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly 290 295 300 Gly Tyr Tyr Asp MetArg Asn Ile Leu Asn Gly Ser Val Val Gln Lys 305 310 315 320 His Pro ThrHis Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro 325 330 335 Gly GluAla Leu Glu Ser Phe Val Gln Gln Trp Phe Lys Pro Leu Ala 340 345 350 TyrAla Leu Val Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr 355 360 365Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser 370 375380 Lys Ile Asp Pro Leu Leu Gln Ala Arg Gln Thr Phe Ala Tyr Gly Thr 385390 395 400 Gln His Asp Tyr Phe Asp His His Asp Ile Ile Gly Trp Thr ArgGlu 405 410 415 Gly Asn Ser Ser His Pro Asn Ser Gly Leu Ala Thr Ile MetSer Asp 420 425 430 Gly Pro Gly Gly Asn Lys Trp Met Tyr Val Gly Lys AsnLys Ala Gly 435 440 445 Gln Val Trp Arg Asp Ile Thr Gly Asn Arg Thr GlyThr Val Thr Ile 450 455 460 Asn Ala Asp Gly Trp Gly Asn Phe Ser Val AsnGly Gly Ser Val Ser 465 470 475 480 Val Trp Val Lys Gln 485 3 1455 DNABacillus sp. CDS (1)..(1455) 3 cat cat aat ggg aca aat ggg acg atg atgcaa tac ttt gaa tgg cac 48 His His Asn Gly Thr Asn Gly Thr Met Met GlnTyr Phe Glu Trp His 1 5 10 15 ttg cct aat gat ggg aat cac tgg aat agatta aga gat gat gct agt 96 Leu Pro Asn Asp Gly Asn His Trp Asn Arg LeuArg Asp Asp Ala Ser 20 25 30 aat cta aga aat aga ggt ata acc gct att tggatt ccg cct gcc tgg 144 Asn Leu Arg Asn Arg Gly Ile Thr Ala Ile Trp IlePro Pro Ala Trp 35 40 45 aaa ggg act tcg caa aat gat gtg ggg tat gga gcctat gat ctt tat 192 Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala TyrAsp Leu Tyr 50 55 60 gat tta ggg gaa ttt aat caa aag ggg acg gtt cgt actaag tat ggg 240 Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr LysTyr Gly 65 70 75 80 aca cgt agt caa ttg gag tct gcc atc cat gct tta aagaat aat ggc 288 Thr Arg Ser Gln Leu Glu Ser Ala Ile His Ala Leu Lys AsnAsn Gly 85 90 95 gtt caa gtt tat ggg gat gta gtg atg aac cat aaa gga ggagct gat 336 Val Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly AlaAsp 100 105 110 gct aca gaa aac gtt ctt gct gtc gag gtg aat cca aat aaccgg aat 384 Ala Thr Glu Asn Val Leu Ala Val Glu Val Asn Pro Asn Asn ArgAsn 115 120 125 caa gaa ata tct ggg gac tac aca att gag gct tgg act aagttt gat 432 Gln Glu Ile Ser Gly Asp Tyr Thr Ile Glu Ala Trp Thr Lys PheAsp 130 135 140 ttt cca ggg agg ggt aat aca tac tca gac ttt aaa tgg cgttgg tat 480 Phe Pro Gly Arg Gly Asn Thr Tyr Ser Asp Phe Lys Trp Arg TrpTyr 145 150 155 160 cat ttc gat ggt gta gat tgg gat caa tca cga caa ttccaa aat cgt 528 His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Gln Phe GlnAsn Arg 165 170 175 atc tac aaa ttc cga ggt gat ggt aag gca tgg gat tgggaa gta gat 576 Ile Tyr Lys Phe Arg Gly Asp Gly Lys Ala Trp Asp Trp GluVal Asp 180 185 190 tcg gaa aat gga aat tat gat tat tta atg tat gca gatgta gat atg 624 Ser Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp ValAsp Met 195 200 205 gat cat ccg gag gta gta aat gag ctt aga aga tgg ggagaa tgg tat 672 Asp His Pro Glu Val Val Asn Glu Leu Arg Arg Trp Gly GluTrp Tyr 210 215 220 aca aat aca tta aat ctt gat gga ttt agg atc gat gcggtg aag cat 720 Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala ValLys His 225 230 235 240 att aaa tat agc ttt aca cgt gat tgg ttg acc catgta aga aac gca 768 Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His ValArg Asn Ala 245 250 255 acg gga aaa gaa atg ttt gct gtt gct gaa ttt tggaaa aat gat tta 816 Thr Gly Lys Glu Met Phe Ala Val Ala Glu Phe Trp LysAsn Asp Leu 260 265 270 ggt gcc ttg gag aac tat tta aat aaa aca aac tggaat cat tct gtc 864 Gly Ala Leu Glu Asn Tyr Leu Asn Lys Thr Asn Trp AsnHis Ser Val 275 280 285 ttt gat gtc ccc ctt cat tat aat ctt tat aac gcgtca aat agt gga 912 Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala SerAsn Ser Gly 290 295 300 ggc aac tat gac atg gca aaa ctt ctt aat gga acggtt gtt caa aag 960 Gly Asn Tyr Asp Met Ala Lys Leu Leu Asn Gly Thr ValVal Gln Lys 305 310 315 320 cat cca atg cat gcc gta act ttt gtg gat aatcac gat tct caa cct 1008 His Pro Met His Ala Val Thr Phe Val Asp Asn HisAsp Ser Gln Pro 325 330 335 ggg gaa tca tta gaa tca ttt gta caa gaa tggttt aag cca ctt gct 1056 Gly Glu Ser Leu Glu Ser Phe Val Gln Glu Trp PheLys Pro Leu Ala 340 345 350 tat gcg ctt att tta aca aga gaa caa ggc tatccc tct gtc ttc tat 1104 Tyr Ala Leu Ile Leu Thr Arg Glu Gln Gly Tyr ProSer Val Phe Tyr 355 360 365 ggt gac tac tat gga att cca aca cat agt gtccca gca atg aaa gcc 1152 Gly Asp Tyr Tyr Gly Ile Pro Thr His Ser Val ProAla Met Lys Ala 370 375 380 aag att gat cca atc tta gag gcg cgt caa aatttt gca tat gga aca 1200 Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Asn PheAla Tyr Gly Thr 385 390 395 400 caa cat gat tat ttt gac cat cat aat ataatc gga tgg aca cgt gaa 1248 Gln His Asp Tyr Phe Asp His His Asn Ile IleGly Trp Thr Arg Glu 405 410 415 gga aat acc acg cat ccc aat tca gga cttgcg act atc atg tcg gat 1296 Gly Asn Thr Thr His Pro Asn Ser Gly Leu AlaThr Ile Met Ser Asp 420 425 430 ggg cca ggg gga gag aaa tgg atg tac gtaggg caa aat aaa gca ggt 1344 Gly Pro Gly Gly Glu Lys Trp Met Tyr Val GlyGln Asn Lys Ala Gly 435 440 445 caa gtt tgg cat gac ata act gga aat aaacca gga aca gtt acg atc 1392 Gln Val Trp His Asp Ile Thr Gly Asn Lys ProGly Thr Val Thr Ile 450 455 460 aat gca gat gga tgg gct aat ttt tca gtaaat gga gga tct gtt tcc 1440 Asn Ala Asp Gly Trp Ala Asn Phe Ser Val AsnGly Gly Ser Val Ser 465 470 475 480 att tgg gtg aaa cga 1455 Ile Trp ValLys Arg 485 4 485 PRT Bacillus sp. 4 His His Asn Gly Thr Asn Gly Thr MetMet Gln Tyr Phe Glu Trp His 1 5 10 15 Leu Pro Asn Asp Gly Asn His TrpAsn Arg Leu Arg Asp Asp Ala Ser 20 25 30 Asn Leu Arg Asn Arg Gly Ile ThrAla Ile Trp Ile Pro Pro Ala Trp 35 40 45 Lys Gly Thr Ser Gln Asn Asp ValGly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60 Asp Leu Gly Glu Phe Asn Gln LysGly Thr Val Arg Thr Lys Tyr Gly 65 70 75 80 Thr Arg Ser Gln Leu Glu SerAla Ile His Ala Leu Lys Asn Asn Gly 85 90 95 Val Gln Val Tyr Gly Asp ValVal Met Asn His Lys Gly Gly Ala Asp 100 105 110 Ala Thr Glu Asn Val LeuAla Val Glu Val Asn Pro Asn Asn Arg Asn 115 120 125 Gln Glu Ile Ser GlyAsp Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp 130 135 140 Phe Pro Gly ArgGly Asn Thr Tyr Ser Asp Phe Lys Trp Arg Trp Tyr 145 150 155 160 His PheAsp Gly Val Asp Trp Asp Gln Ser Arg Gln Phe Gln Asn Arg 165 170 175 IleTyr Lys Phe Arg Gly Asp Gly Lys Ala Trp Asp Trp Glu Val Asp 180 185 190Ser Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met 195 200205 Asp His Pro Glu Val Val Asn Glu Leu Arg Arg Trp Gly Glu Trp Tyr 210215 220 Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His225 230 235 240 Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val ArgAsn Ala 245 250 255 Thr Gly Lys Glu Met Phe Ala Val Ala Glu Phe Trp LysAsn Asp Leu 260 265 270 Gly Ala Leu Glu Asn Tyr Leu Asn Lys Thr Asn TrpAsn His Ser Val 275 280 285 Phe Asp Val Pro Leu His Tyr Asn Leu Tyr AsnAla Ser Asn Ser Gly 290 295 300 Gly Asn Tyr Asp Met Ala Lys Leu Leu AsnGly Thr Val Val Gln Lys 305 310 315 320 His Pro Met His Ala Val Thr PheVal Asp Asn His Asp Ser Gln Pro 325 330 335 Gly Glu Ser Leu Glu Ser PheVal Gln Glu Trp Phe Lys Pro Leu Ala 340 345 350 Tyr Ala Leu Ile Leu ThrArg Glu Gln Gly Tyr Pro Ser Val Phe Tyr 355 360 365 Gly Asp Tyr Tyr GlyIle Pro Thr His Ser Val Pro Ala Met Lys Ala 370 375 380 Lys Ile Asp ProIle Leu Glu Ala Arg Gln Asn Phe Ala Tyr Gly Thr 385 390 395 400 Gln HisAsp Tyr Phe Asp His His Asn Ile Ile Gly Trp Thr Arg Glu 405 410 415 GlyAsn Thr Thr His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp 420 425 430Gly Pro Gly Gly Glu Lys Trp Met Tyr Val Gly Gln Asn Lys Ala Gly 435 440445 Gln Val Trp His Asp Ile Thr Gly Asn Lys Pro Gly Thr Val Thr Ile 450455 460 Asn Ala Asp Gly Trp Ala Asn Phe Ser Val Asn Gly Gly Ser Val Ser465 470 475 480 Ile Trp Val Lys Arg 485 5 1548 DNA Bacillusstearothermophilus CDS (1)..(1548) 5 gcc gca ccg ttt aac ggc acc atg atgcag tat ttt gaa tgg tac ttg 48 Ala Ala Pro Phe Asn Gly Thr Met Met GlnTyr Phe Glu Trp Tyr Leu 1 5 10 15 ccg gat gat ggc acg tta tgg acc aaagtg gcc aat gaa gcc aac aac 96 Pro Asp Asp Gly Thr Leu Trp Thr Lys ValAla Asn Glu Ala Asn Asn 20 25 30 tta tcc agc ctt ggc atc acc gct ctt tggctg ccg ccc gct tac aaa 144 Leu Ser Ser Leu Gly Ile Thr Ala Leu Trp LeuPro Pro Ala Tyr Lys 35 40 45 gga aca agc cgc agc gac gta ggg tac gga gtatac gac ttg tat gac 192 Gly Thr Ser Arg Ser Asp Val Gly Tyr Gly Val TyrAsp Leu Tyr Asp 50 55 60 ctc ggc gaa ttc aat caa aaa ggg acc gtc cgc acaaaa tac gga aca 240 Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr LysTyr Gly Thr 65 70 75 80 aaa gct caa tat ctt caa gcc att caa gcc gcc cacgcc gct gga atg 288 Lys Ala Gln Tyr Leu Gln Ala Ile Gln Ala Ala His AlaAla Gly Met 85 90 95 caa gtg tac gcc gat gtc gtg ttc gac cat aaa ggc ggcgct gac ggc 336 Gln Val Tyr Ala Asp Val Val Phe Asp His Lys Gly Gly AlaAsp Gly 100 105 110 acg gaa tgg gtg gac gcc gtc gaa gtc aat ccg tcc gaccgc aac caa 384 Thr Glu Trp Val Asp Ala Val Glu Val Asn Pro Ser Asp ArgAsn Gln 115 120 125 gaa atc tcg ggc acc tat caa atc caa gca tgg acg aaattt gat ttt 432 Glu Ile Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys PheAsp Phe 130 135 140 ccc ggg cgg ggc aac acc tac tcc agc ttt aag tgg cgctgg tac cat 480 Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg TrpTyr His 145 150 155 160 ttt gac ggc gtt gat tgg gac gaa agc cga aaa ttgagc cgc att tac 528 Phe Asp Gly Val Asp Trp Asp Glu Ser Arg Lys Leu SerArg Ile Tyr 165 170 175 aaa ttc cgc ggc atc ggc aaa gcg tgg gat tgg gaagta gac acg gaa 576 Lys Phe Arg Gly Ile Gly Lys Ala Trp Asp Trp Glu ValAsp Thr Glu 180 185 190 aac gga aac tat gac tac tta atg tat gcc gac cttgat atg gat cat 624 Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu AspMet Asp His 195 200 205 ccc gaa gtc gtg acc gag ctg aaa aac tgg ggg aaatgg tat gtc aac 672 Pro Glu Val Val Thr Glu Leu Lys Asn Trp Gly Lys TrpTyr Val Asn 210 215 220 aca acg aac att gat ggg ttc cgg ctt gat gcc gtcaag cat att aag 720 Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val LysHis Ile Lys 225 230 235 240 ttc agt ttt ttt cct gat tgg ttg tcg tat gtgcgt tct cag act ggc 768 Phe Ser Phe Phe Pro Asp Trp Leu Ser Tyr Val ArgSer Gln Thr Gly 245 250 255 aag ccg cta ttt acc gtc ggg gaa tat tgg agctat gac atc aac aag 816 Lys Pro Leu Phe Thr Val Gly Glu Tyr Trp Ser TyrAsp Ile Asn Lys 260 265 270 ttg cac aat tac att acg aaa aca gac gga acgatg tct ttg ttt gat 864 Leu His Asn Tyr Ile Thr Lys Thr Asp Gly Thr MetSer Leu Phe Asp 275 280 285 gcc ccg tta cac aac aaa ttt tat acc gct tccaaa tca ggg ggc gca 912 Ala Pro Leu His Asn Lys Phe Tyr Thr Ala Ser LysSer Gly Gly Ala 290 295 300 ttt gat atg cgc acg tta atg acc aat act ctcatg aaa gat caa ccg 960 Phe Asp Met Arg Thr Leu Met Thr Asn Thr Leu MetLys Asp Gln Pro 305 310 315 320 aca ttg gcc gtc acc ttc gtt gat aat catgac acc gaa ccc ggc caa 1008 Thr Leu Ala Val Thr Phe Val Asp Asn His AspThr Glu Pro Gly Gln 325 330 335 gcg ctg cag tca tgg gtc gac cca tgg ttcaaa ccg ttg gct tac gcc 1056 Ala Leu Gln Ser Trp Val Asp Pro Trp Phe LysPro Leu Ala Tyr Ala 340 345 350 ttt att cta act cgg cag gaa gga tac ccgtgc gtc ttt tat ggt gac 1104 Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro CysVal Phe Tyr Gly Asp 355 360 365 tat tat ggc att cca caa tat aac att ccttcg ctg aaa agc aaa atc 1152 Tyr Tyr Gly Ile Pro Gln Tyr Asn Ile Pro SerLeu Lys Ser Lys Ile 370 375 380 gat ccg ctc ctc atc gcg cgc agg gat tatgct tac gga acg caa cat 1200 Asp Pro Leu Leu Ile Ala Arg Arg Asp Tyr AlaTyr Gly Thr Gln His 385 390 395 400 gat tat ctt gat cac tcc gac atc atcggg tgg aca agg gaa ggg ggc 1248 Asp Tyr Leu Asp His Ser Asp Ile Ile GlyTrp Thr Arg Glu Gly Gly 405 410 415 act gaa aaa cca gga tcc gga ctg gccgca ctg atc acc gat ggg ccg 1296 Thr Glu Lys Pro Gly Ser Gly Leu Ala AlaLeu Ile Thr Asp Gly Pro 420 425 430 gga gga agc aaa tgg atg tac gtt ggcaaa caa cac gct gga aaa gtg 1344 Gly Gly Ser Lys Trp Met Tyr Val Gly LysGln His Ala Gly Lys Val 435 440 445 ttc tat gac ctt acc ggc aac cgg agtgac acc gtc acc atc aac agt 1392 Phe Tyr Asp Leu Thr Gly Asn Arg Ser AspThr Val Thr Ile Asn Ser 450 455 460 gat gga tgg ggg gaa ttc aaa gtc aatggc ggt tcg gtt tcg gtt tgg 1440 Asp Gly Trp Gly Glu Phe Lys Val Asn GlyGly Ser Val Ser Val Trp 465 470 475 480 gtt cct aga aaa acg acc gtt tctacc atc gct cgg ccg atc aca acc 1488 Val Pro Arg Lys Thr Thr Val Ser ThrIle Ala Arg Pro Ile Thr Thr 485 490 495 cga ccg tgg act ggt gaa ttc gtccgt tgg acc gaa cca cgg ttg gtg 1536 Arg Pro Trp Thr Gly Glu Phe Val ArgTrp Thr Glu Pro Arg Leu Val 500 505 510 gca tgg cct tga 1548 Ala Trp Pro515 6 515 PRT Bacillus stearothermophilus 6 Ala Ala Pro Phe Asn Gly ThrMet Met Gln Tyr Phe Glu Trp Tyr Leu 1 5 10 15 Pro Asp Asp Gly Thr LeuTrp Thr Lys Val Ala Asn Glu Ala Asn Asn 20 25 30 Leu Ser Ser Leu Gly IleThr Ala Leu Trp Leu Pro Pro Ala Tyr Lys 35 40 45 Gly Thr Ser Arg Ser AspVal Gly Tyr Gly Val Tyr Asp Leu Tyr Asp 50 55 60 Leu Gly Glu Phe Asn GlnLys Gly Thr Val Arg Thr Lys Tyr Gly Thr 65 70 75 80 Lys Ala Gln Tyr LeuGln Ala Ile Gln Ala Ala His Ala Ala Gly Met 85 90 95 Gln Val Tyr Ala AspVal Val Phe Asp His Lys Gly Gly Ala Asp Gly 100 105 110 Thr Glu Trp ValAsp Ala Val Glu Val Asn Pro Ser Asp Arg Asn Gln 115 120 125 Glu Ile SerGly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp Phe 130 135 140 Pro GlyArg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr His 145 150 155 160Phe Asp Gly Val Asp Trp Asp Glu Ser Arg Lys Leu Ser Arg Ile Tyr 165 170175 Lys Phe Arg Gly Ile Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu 180185 190 Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met Asp His195 200 205 Pro Glu Val Val Thr Glu Leu Lys Asn Trp Gly Lys Trp Tyr ValAsn 210 215 220 Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys HisIle Lys 225 230 235 240 Phe Ser Phe Phe Pro Asp Trp Leu Ser Tyr Val ArgSer Gln Thr Gly 245 250 255 Lys Pro Leu Phe Thr Val Gly Glu Tyr Trp SerTyr Asp Ile Asn Lys 260 265 270 Leu His Asn Tyr Ile Thr Lys Thr Asp GlyThr Met Ser Leu Phe Asp 275 280 285 Ala Pro Leu His Asn Lys Phe Tyr ThrAla Ser Lys Ser Gly Gly Ala 290 295 300 Phe Asp Met Arg Thr Leu Met ThrAsn Thr Leu Met Lys Asp Gln Pro 305 310 315 320 Thr Leu Ala Val Thr PheVal Asp Asn His Asp Thr Glu Pro Gly Gln 325 330 335 Ala Leu Gln Ser TrpVal Asp Pro Trp Phe Lys Pro Leu Ala Tyr Ala 340 345 350 Phe Ile Leu ThrArg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly Asp 355 360 365 Tyr Tyr GlyIle Pro Gln Tyr Asn Ile Pro Ser Leu Lys Ser Lys Ile 370 375 380 Asp ProLeu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln His 385 390 395 400Asp Tyr Leu Asp His Ser Asp Ile Ile Gly Trp Thr Arg Glu Gly Gly 405 410415 Thr Glu Lys Pro Gly Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro 420425 430 Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Gln His Ala Gly Lys Val435 440 445 Phe Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile AsnSer 450 455 460 Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val SerVal Trp 465 470 475 480 Val Pro Arg Lys Thr Thr Val Ser Thr Ile Ala ArgPro Ile Thr Thr 485 490 495 Arg Pro Trp Thr Gly Glu Phe Val Arg Trp ThrGlu Pro Arg Leu Val 500 505 510 Ala Trp Pro 515 7 1920 DNA Bacilluslicheniformis CDS (421)..(1872) 7 cggaagattg gaagtacaaa aataagcaaaagattgtcaa tcatgtcatg agccatgcgg 60 gagacggaaa aatcgtctta atgcacgatatttatgcaac gttcgcagat gctgctgaag 120 agattattaa aaagctgaaa gcaaaaggctatcaattggt aactgtatct cagcttgaag 180 aagtgaagaa gcagagaggc tattgaataaatgagtagaa gcgccatatc ggcgcttttc 240 ttttggaaga aaatataggg aaaatggtacttgttaaaaa ttcggaatat ttatacaaca 300 tcatatgttt cacattgaaa ggggaggagaatcatgaaac aacaaaaacg gctttacgcc 360 cgattgctga cgctgttatt tgcgctcatcttcttgctgc ctcattctgc agcagcggcg 420 gca aat ctt aat ggg acg ctg atg cagtat ttt gaa tgg tac atg ccc 468 Ala Asn Leu Asn Gly Thr Leu Met Gln TyrPhe Glu Trp Tyr Met Pro 1 5 10 15 aat gac ggc caa cat tgg agg cgt ttgcaa aac gac tcg gca tat ttg 516 Asn Asp Gly Gln His Trp Arg Arg Leu GlnAsn Asp Ser Ala Tyr Leu 20 25 30 gct gaa cac ggt att act gcc gtc tgg attccc ccg gca tat aag gga 564 Ala Glu His Gly Ile Thr Ala Val Trp Ile ProPro Ala Tyr Lys Gly 35 40 45 acg agc caa gcg gat gtg ggc tac ggt gct tacgac ctt tat gat tta 612 Thr Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr AspLeu Tyr Asp Leu 50 55 60 ggg gag ttt cat caa aaa ggg acg gtt cgg aca aagtac ggc aca aaa 660 Gly Glu Phe His Gln Lys Gly Thr Val Arg Thr Lys TyrGly Thr Lys 65 70 75 80 gga gag ctg caa tct gcg atc aaa agt ctt cat tcccgc gac att aac 708 Gly Glu Leu Gln Ser Ala Ile Lys Ser Leu His Ser ArgAsp Ile Asn 85 90 95 gtt tac ggg gat gtg gtc atc aac cac aaa ggc ggc gctgat gcg acc 756 Val Tyr Gly Asp Val Val Ile Asn His Lys Gly Gly Ala AspAla Thr 100 105 110 gaa gat gta acc gcg gtt gaa gtc gat ccc gct gac cgcaac cgc gta 804 Glu Asp Val Thr Ala Val Glu Val Asp Pro Ala Asp Arg AsnArg Val 115 120 125 att tca gga gaa cac cta att aaa gcc tgg aca cat tttcat ttt ccg 852 Ile Ser Gly Glu His Leu Ile Lys Ala Trp Thr His Phe HisPhe Pro 130 135 140 ggg cgc ggc agc aca tac agc gat ttt aaa tgg cat tggtac cat ttt 900 Gly Arg Gly Ser Thr Tyr Ser Asp Phe Lys Trp His Trp TyrHis Phe 145 150 155 160 gac gga acc gat tgg gac gag tcc cga aag ctg aaccgc atc tat aag 948 Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn ArgIle Tyr Lys 165 170 175 ttt caa gga aag gct tgg gat tgg gaa gtt tcc aatgaa aac ggc aac 996 Phe Gln Gly Lys Ala Trp Asp Trp Glu Val Ser Asn GluAsn Gly Asn 180 185 190 tat gat tat ttg atg tat gcc gac atc gat tat gaccat cct gat gtc 1044 Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Tyr Asp HisPro Asp Val 195 200 205 gca gca gaa att aag aga tgg ggc act tgg tat gccaat gaa ctg caa 1092 Ala Ala Glu Ile Lys Arg Trp Gly Thr Trp Tyr Ala AsnGlu Leu Gln 210 215 220 ttg gac ggt ttc cgt ctt gat gct gtc aaa cac attaaa ttt tct ttt 1140 Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile LysPhe Ser Phe 225 230 235 240 ttg cgg gat tgg gtt aat cat gtc agg gaa aaaacg ggg aag gaa atg 1188 Leu Arg Asp Trp Val Asn His Val Arg Glu Lys ThrGly Lys Glu Met 245 250 255 ttt acg gta gct gaa tat tgg cag aat gac ttgggc gcg ctg gaa aac 1236 Phe Thr Val Ala Glu Tyr Trp Gln Asn Asp Leu GlyAla Leu Glu Asn 260 265 270 tat ttg aac aaa aca aat ttt aat cat tca gtgttt gac gtg ccg ctt 1284 Tyr Leu Asn Lys Thr Asn Phe Asn His Ser Val PheAsp Val Pro Leu 275 280 285 cat tat cag ttc cat gct gca tcg aca cag ggaggc ggc tat gat atg 1332 His Tyr Gln Phe His Ala Ala Ser Thr Gln Gly GlyGly Tyr Asp Met 290 295 300 agg aaa ttg ctg aac ggt acg gtc gtt tcc aagcat ccg ttg aaa tcg 1380 Arg Lys Leu Leu Asn Gly Thr Val Val Ser Lys HisPro Leu Lys Ser 305 310 315 320 gtt aca ttt gtc gat aac cat gat aca cagccg ggg caa tcg ctt gag 1428 Val Thr Phe Val Asp Asn His Asp Thr Gln ProGly Gln Ser Leu Glu 325 330 335 tcg act gtc caa aca tgg ttt aag ccg cttgct tac gct ttt att ctc 1476 Ser Thr Val Gln Thr Trp Phe Lys Pro Leu AlaTyr Ala Phe Ile Leu 340 345 350 aca agg gaa tct gga tac cct cag gtt ttctac ggg gat atg tac ggg 1524 Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe TyrGly Asp Met Tyr Gly 355 360 365 acg aaa gga gac tcc cag cgc gaa att cctgcc ttg aaa cac aaa att 1572 Thr Lys Gly Asp Ser Gln Arg Glu Ile Pro AlaLeu Lys His Lys Ile 370 375 380 gaa ccg atc tta aaa gcg aga aaa cag tatgcg tac gga gca cag cat 1620 Glu Pro Ile Leu Lys Ala Arg Lys Gln Tyr AlaTyr Gly Ala Gln His 385 390 395 400 gat tat ttc gac cac cat gac att gtcggc tgg aca agg gaa ggc gac 1668 Asp Tyr Phe Asp His His Asp Ile Val GlyTrp Thr Arg Glu Gly Asp 405 410 415 agc tcg gtt gca aat tca ggt ttg gcggca tta ata aca gac gga ccc 1716 Ser Ser Val Ala Asn Ser Gly Leu Ala AlaLeu Ile Thr Asp Gly Pro 420 425 430 ggt ggg gca aag cga atg tat gtc ggccgg caa aac gcc ggt gag aca 1764 Gly Gly Ala Lys Arg Met Tyr Val Gly ArgGln Asn Ala Gly Glu Thr 435 440 445 tgg cat gac att acc gga aac cgt tcggag ccg gtt gtc atc aat tcg 1812 Trp His Asp Ile Thr Gly Asn Arg Ser GluPro Val Val Ile Asn Ser 450 455 460 gaa ggc tgg gga gag ttt cac gta aacggc ggg tcg gtt tca att tat 1860 Glu Gly Trp Gly Glu Phe His Val Asn GlyGly Ser Val Ser Ile Tyr 465 470 475 480 gtt caa aga tag aagagcagagaggacggatt tcctgaagga aatccgtttt 1912 Val Gln Arg tttatttt 1920 8 483PRT Bacillus licheniformis 8 Ala Asn Leu Asn Gly Thr Leu Met Gln Tyr PheGlu Trp Tyr Met Pro 1 5 10 15 Asn Asp Gly Gln His Trp Arg Arg Leu GlnAsn Asp Ser Ala Tyr Leu 20 25 30 Ala Glu His Gly Ile Thr Ala Val Trp IlePro Pro Ala Tyr Lys Gly 35 40 45 Thr Ser Gln Ala Asp Val Gly Tyr Gly AlaTyr Asp Leu Tyr Asp Leu 50 55 60 Gly Glu Phe His Gln Lys Gly Thr Val ArgThr Lys Tyr Gly Thr Lys 65 70 75 80 Gly Glu Leu Gln Ser Ala Ile Lys SerLeu His Ser Arg Asp Ile Asn 85 90 95 Val Tyr Gly Asp Val Val Ile Asn HisLys Gly Gly Ala Asp Ala Thr 100 105 110 Glu Asp Val Thr Ala Val Glu ValAsp Pro Ala Asp Arg Asn Arg Val 115 120 125 Ile Ser Gly Glu His Leu IleLys Ala Trp Thr His Phe His Phe Pro 130 135 140 Gly Arg Gly Ser Thr TyrSer Asp Phe Lys Trp His Trp Tyr His Phe 145 150 155 160 Asp Gly Thr AspTrp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys 165 170 175 Phe Gln GlyLys Ala Trp Asp Trp Glu Val Ser Asn Glu Asn Gly Asn 180 185 190 Tyr AspTyr Leu Met Tyr Ala Asp Ile Asp Tyr Asp His Pro Asp Val 195 200 205 AlaAla Glu Ile Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu Gln 210 215 220Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe 225 230235 240 Leu Arg Asp Trp Val Asn His Val Arg Glu Lys Thr Gly Lys Glu Met245 250 255 Phe Thr Val Ala Glu Tyr Trp Gln Asn Asp Leu Gly Ala Leu GluAsn 260 265 270 Tyr Leu Asn Lys Thr Asn Phe Asn His Ser Val Phe Asp ValPro Leu 275 280 285 His Tyr Gln Phe His Ala Ala Ser Thr Gln Gly Gly GlyTyr Asp Met 290 295 300 Arg Lys Leu Leu Asn Gly Thr Val Val Ser Lys HisPro Leu Lys Ser 305 310 315 320 Val Thr Phe Val Asp Asn His Asp Thr GlnPro Gly Gln Ser Leu Glu 325 330 335 Ser Thr Val Gln Thr Trp Phe Lys ProLeu Ala Tyr Ala Phe Ile Leu 340 345 350 Thr Arg Glu Ser Gly Tyr Pro GlnVal Phe Tyr Gly Asp Met Tyr Gly 355 360 365 Thr Lys Gly Asp Ser Gln ArgGlu Ile Pro Ala Leu Lys His Lys Ile 370 375 380 Glu Pro Ile Leu Lys AlaArg Lys Gln Tyr Ala Tyr Gly Ala Gln His 385 390 395 400 Asp Tyr Phe AspHis His Asp Ile Val Gly Trp Thr Arg Glu Gly Asp 405 410 415 Ser Ser ValAla Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro 420 425 430 Gly GlyAla Lys Arg Met Tyr Val Gly Arg Gln Asn Ala Gly Glu Thr 435 440 445 TrpHis Asp Ile Thr Gly Asn Arg Ser Glu Pro Val Val Ile Asn Ser 450 455 460Glu Gly Trp Gly Glu Phe His Val Asn Gly Gly Ser Val Ser Ile Tyr 465 470475 480 Val Gln Arg 9 2084 DNA Bacillus amyloliquefaciens CDS(343)..(1794) 9 gccccgcaca tacgaaaaga ctggctgaaa acattgagcc tttgatgactgatgatttgg 60 ctgaagaagt ggatcgattg tttgagaaaa gaagaagacc ataaaaataccttgtctgtc 120 atcagacagg gtatttttta tgctgtccag actgtccgct gtgtaaaaataaggaataaa 180 ggggggttgt tattatttta ctgatatgta aaatataatt tgtataagaaaatgagaggg 240 agaggaaaca tgattcaaaa acgaaagcgg acagtttcgt tcagacttgtgcttatgtgc 300 acgctgttat ttgtcagttt gccgattaca aaaacatcag cc gta aatggc acg 354 Val Asn Gly Thr 1 ctg atg cag tat ttt gaa tgg tat acg ccgaac gac ggc cag cat tgg 402 Leu Met Gln Tyr Phe Glu Trp Tyr Thr Pro AsnAsp Gly Gln His Trp 5 10 15 20 aaa cga ttg cag aat gat gcg gaa cat ttatcg gat atc gga atc act 450 Lys Arg Leu Gln Asn Asp Ala Glu His Leu SerAsp Ile Gly Ile Thr 25 30 35 gcc gtc tgg att cct ccc gca tac aaa gga ttgagc caa tcc gat aac 498 Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Leu SerGln Ser Asp Asn 40 45 50 gga tac gga cct tat gat ttg tat gat tta gga gaattc cag caa aaa 546 Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu Gly Glu PheGln Gln Lys 55 60 65 ggg acg gtc aga acg aaa tac ggc aca aaa tca gag cttcaa gat gcg 594 Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Ser Glu Leu GlnAsp Ala 70 75 80 atc ggc tca ctg cat tcc cgg aac gtc caa gta tac gga gatgtg gtt 642 Ile Gly Ser Leu His Ser Arg Asn Val Gln Val Tyr Gly Asp ValVal 85 90 95 100 ttg aat cat aag gct ggt gct gat gca aca gaa gat gta actgcc gtc 690 Leu Asn His Lys Ala Gly Ala Asp Ala Thr Glu Asp Val Thr AlaVal 105 110 115 gaa gtc aat ccg gcc aat aga aat cag gaa act tcg gag gaatat caa 738 Glu Val Asn Pro Ala Asn Arg Asn Gln Glu Thr Ser Glu Glu TyrGln 120 125 130 atc aaa gcg tgg acg gat ttt cgt ttt ccg ggc cgt gga aacacg tac 786 Ile Lys Ala Trp Thr Asp Phe Arg Phe Pro Gly Arg Gly Asn ThrTyr 135 140 145 agt gat ttt aaa tgg cat tgg tat cat ttc gac gga gcg gactgg gat 834 Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly Ala Asp TrpAsp 150 155 160 gaa tcc cgg aag atc agc cgc atc ttt aag ttt cgt ggg gaagga aaa 882 Glu Ser Arg Lys Ile Ser Arg Ile Phe Lys Phe Arg Gly Glu GlyLys 165 170 175 180 gcg tgg gat tgg gaa gta tca agt gaa aac ggc aac tatgac tat tta 930 Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly Asn Tyr AspTyr Leu 185 190 195 atg tat gct gat gtt gac tac gac cac cct gat gtc gtggca gag aca 978 Met Tyr Ala Asp Val Asp Tyr Asp His Pro Asp Val Val AlaGlu Thr 200 205 210 aaa aaa tgg ggt atc tgg tat gcg aat gaa ctg tca ttagac ggc ttc 1026 Lys Lys Trp Gly Ile Trp Tyr Ala Asn Glu Leu Ser Leu AspGly Phe 215 220 225 cgt att gat gcc gcc aaa cat att aaa ttt tca ttt ctgcgt gat tgg 1074 Arg Ile Asp Ala Ala Lys His Ile Lys Phe Ser Phe Leu ArgAsp Trp 230 235 240 gtt cag gcg gtc aga cag gcg acg gga aaa gaa atg tttacg gtt gcg 1122 Val Gln Ala Val Arg Gln Ala Thr Gly Lys Glu Met Phe ThrVal Ala 245 250 255 260 gag tat tgg cag aat aat gcc ggg aaa ctc gaa aactac ttg aat aaa 1170 Glu Tyr Trp Gln Asn Asn Ala Gly Lys Leu Glu Asn TyrLeu Asn Lys 265 270 275 aca agc ttt aat caa tcc gtg ttt gat gtt ccg cttcat ttc aat tta 1218 Thr Ser Phe Asn Gln Ser Val Phe Asp Val Pro Leu HisPhe Asn Leu 280 285 290 cag gcg gct tcc tca caa gga ggc gga tat gat atgagg cgt ttg ctg 1266 Gln Ala Ala Ser Ser Gln Gly Gly Gly Tyr Asp Met ArgArg Leu Leu 295 300 305 gac ggt acc gtt gtg tcc agg cat ccg gaa aag gcggtt aca ttt gtt 1314 Asp Gly Thr Val Val Ser Arg His Pro Glu Lys Ala ValThr Phe Val 310 315 320 gaa aat cat gac aca cag ccg gga cag tca ttg gaatcg aca gtc caa 1362 Glu Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu SerThr Val Gln 325 330 335 340 act tgg ttt aaa ccg ctt gca tac gcc ttt attttg aca aga gaa tcc 1410 Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile LeuThr Arg Glu Ser 345 350 355 ggt tat cct cag gtg ttc tat ggg gat atg tacggg aca aaa ggg aca 1458 Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr GlyThr Lys Gly Thr 360 365 370 tcg cca aag gaa att ccc tca ctg aaa gat aatata gag ccg att tta 1506 Ser Pro Lys Glu Ile Pro Ser Leu Lys Asp Asn IleGlu Pro Ile Leu 375 380 385 aaa gcg cgt aag gag tac gca tac ggg ccc cagcac gat tat att gac 1554 Lys Ala Arg Lys Glu Tyr Ala Tyr Gly Pro Gln HisAsp Tyr Ile Asp 390 395 400 cac ccg gat gtg atc gga tgg acg agg gaa ggtgac agc tcc gcc gcc 1602 His Pro Asp Val Ile Gly Trp Thr Arg Glu Gly AspSer Ser Ala Ala 405 410 415 420 aaa tca ggt ttg gcc gct tta atc acg gacgga ccc ggc gga tca aag 1650 Lys Ser Gly Leu Ala Ala Leu Ile Thr Asp GlyPro Gly Gly Ser Lys 425 430 435 cgg atg tat gcc ggc ctg aaa aat gcc ggcgag aca tgg tat gac ata 1698 Arg Met Tyr Ala Gly Leu Lys Asn Ala Gly GluThr Trp Tyr Asp Ile 440 445 450 acg ggc aac cgt tca gat act gta aaa atcgga tct gac ggc tgg gga 1746 Thr Gly Asn Arg Ser Asp Thr Val Lys Ile GlySer Asp Gly Trp Gly 455 460 465 gag ttt cat gta aac gat ggg tcc gtc tccatt tat gtt cag aaa taa 1794 Glu Phe His Val Asn Asp Gly Ser Val Ser IleTyr Val Gln Lys 470 475 480 ggtaataaaa aaacacctcc aagctgagtg cgggtatcagcttggaggtg cgtttatttt 1854 ttcagccgta tgacaaggtc ggcatcaggt gtgacaaatacggtatgctg gctgtcatag 1914 gtgacaaatc cgggttttgc gccgtttggc tttttcacatgtctgatttt tgtataatca 1974 acaggcacgg agccggaatc tttcgccttg gaaaaataagcggcgatcgt agctgcttcc 2034 aatatggatt gttcatcggg atcgctgctt ttaatcacaacgtgggatcc 2084 10 483 PRT Bacillus amyloliquefaciens 10 Val Asn Gly ThrLeu Met Gln Tyr Phe Glu Trp Tyr Thr Pro Asn Asp 1 5 10 15 Gly Gln HisTrp Lys Arg Leu Gln Asn Asp Ala Glu His Leu Ser Asp 20 25 30 Ile Gly IleThr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Leu Ser 35 40 45 Gln Ser AspAsn Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu Gly Glu 50 55 60 Phe Gln GlnLys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Ser Glu 65 70 75 80 Leu GlnAsp Ala Ile Gly Ser Leu His Ser Arg Asn Val Gln Val Tyr 85 90 95 Gly AspVal Val Leu Asn His Lys Ala Gly Ala Asp Ala Thr Glu Asp 100 105 110 ValThr Ala Val Glu Val Asn Pro Ala Asn Arg Asn Gln Glu Thr Ser 115 120 125Glu Glu Tyr Gln Ile Lys Ala Trp Thr Asp Phe Arg Phe Pro Gly Arg 130 135140 Gly Asn Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly 145150 155 160 Ala Asp Trp Asp Glu Ser Arg Lys Ile Ser Arg Ile Phe Lys PheArg 165 170 175 Gly Glu Gly Lys Ala Trp Asp Trp Glu Val Ser Ser Glu AsnGly Asn 180 185 190 Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Tyr Asp HisPro Asp Val 195 200 205 Val Ala Glu Thr Lys Lys Trp Gly Ile Trp Tyr AlaAsn Glu Leu Ser 210 215 220 Leu Asp Gly Phe Arg Ile Asp Ala Ala Lys HisIle Lys Phe Ser Phe 225 230 235 240 Leu Arg Asp Trp Val Gln Ala Val ArgGln Ala Thr Gly Lys Glu Met 245 250 255 Phe Thr Val Ala Glu Tyr Trp GlnAsn Asn Ala Gly Lys Leu Glu Asn 260 265 270 Tyr Leu Asn Lys Thr Ser PheAsn Gln Ser Val Phe Asp Val Pro Leu 275 280 285 His Phe Asn Leu Gln AlaAla Ser Ser Gln Gly Gly Gly Tyr Asp Met 290 295 300 Arg Arg Leu Leu AspGly Thr Val Val Ser Arg His Pro Glu Lys Ala 305 310 315 320 Val Thr PheVal Glu Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu 325 330 335 Ser ThrVal Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu 340 345 350 ThrArg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly 355 360 365Thr Lys Gly Thr Ser Pro Lys Glu Ile Pro Ser Leu Lys Asp Asn Ile 370 375380 Glu Pro Ile Leu Lys Ala Arg Lys Glu Tyr Ala Tyr Gly Pro Gln His 385390 395 400 Asp Tyr Ile Asp His Pro Asp Val Ile Gly Trp Thr Arg Glu GlyAsp 405 410 415 Ser Ser Ala Ala Lys Ser Gly Leu Ala Ala Leu Ile Thr AspGly Pro 420 425 430 Gly Gly Ser Lys Arg Met Tyr Ala Gly Leu Lys Asn AlaGly Glu Thr 435 440 445 Trp Tyr Asp Ile Thr Gly Asn Arg Ser Asp Thr ValLys Ile Gly Ser 450 455 460 Asp Gly Trp Gly Glu Phe His Val Asn Asp GlySer Val Ser Ile Tyr 465 470 475 480 Val Gln Lys 11 1458 DNA Bacillus sp.CDS (1)..(1458) 11 cac cat aat ggt acg aac ggc aca atg atg cag tac tttgaa tgg tat 48 His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe GluTrp Tyr 1 5 10 15 cta cca aat gac gga aac cat tgg aat aga tta agg tctgat gca agt 96 Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Ser AspAla Ser 20 25 30 aac cta aaa gat aaa ggg atc tca gcg gtt tgg att cct cctgca tgg 144 Asn Leu Lys Asp Lys Gly Ile Ser Ala Val Trp Ile Pro Pro AlaTrp 35 40 45 aag ggt gcc tct caa aat gat gtg ggg tat ggt gct tat gat ctgtat 192 Lys Gly Ala Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr50 55 60 gat tta gga gaa ttc aat caa aaa gga acc att cgt aca aaa tat gga240 Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr Gly 6570 75 80 acg cgc aat cag tta caa gct gca gtt aac gcc ttg aaa agt aat gga288 Thr Arg Asn Gln Leu Gln Ala Ala Val Asn Ala Leu Lys Ser Asn Gly 8590 95 att caa gtg tat ggc gat gtt gta atg aat cat aaa ggg gga gca gac336 Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100105 110 gct acc gaa atg gtt agg gca gtt gaa gta aac ccg aat aat aga aat384 Ala Thr Glu Met Val Arg Ala Val Glu Val Asn Pro Asn Asn Arg Asn 115120 125 caa gaa gtg tcc ggt gaa tat aca att gag gct tgg aca aag ttt gac432 Gln Glu Val Ser Gly Glu Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp 130135 140 ttt cca gga cga ggt aat act cat tca aac ttc aaa tgg aga tgg tat480 Phe Pro Gly Arg Gly Asn Thr His Ser Asn Phe Lys Trp Arg Trp Tyr 145150 155 160 cac ttt gat gga gta gat tgg gat cag tca cgt aag ctg aac aatcga 528 His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Lys Leu Asn Asn Arg165 170 175 att tat aaa ttt aga ggt gat gga aaa ggg tgg gat tgg gaa gtcgat 576 Ile Tyr Lys Phe Arg Gly Asp Gly Lys Gly Trp Asp Trp Glu Val Asp180 185 190 aca gaa aac ggt aac tat gat tac cta atg tat gca gat att gacatg 624 Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met195 200 205 gat cac cca gag gta gtg aat gag cta aga aat tgg ggt gtt tggtat 672 Asp His Pro Glu Val Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr210 215 220 acg aat aca tta ggc ctt gat ggt ttt aga ata gat gca gta aaacat 720 Thr Asn Thr Leu Gly Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His225 230 235 240 ata aaa tac agc ttt act cgt gat tgg att aat cat gtt agaagt gca 768 Ile Lys Tyr Ser Phe Thr Arg Asp Trp Ile Asn His Val Arg SerAla 245 250 255 act ggc aaa aat atg ttt gcg gtt gcg gaa ttt tgg aaa aatgat tta 816 Thr Gly Lys Asn Met Phe Ala Val Ala Glu Phe Trp Lys Asn AspLeu 260 265 270 ggt gct att gaa aac tat tta aac aaa aca aac tgg aac cattca gtc 864 Gly Ala Ile Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His SerVal 275 280 285 ttt gat gtt ccg ctg cac tat aac ctc tat aat gct tca aaaagc gga 912 Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Lys SerGly 290 295 300 ggg aat tat gat atg agg caa ata ttt aat ggt aca gtc gtgcaa aga 960 Gly Asn Tyr Asp Met Arg Gln Ile Phe Asn Gly Thr Val Val GlnArg 305 310 315 320 cat cca atg cat gct gtt aca ttt gtt gat aat cat gattcg caa cct 1008 His Pro Met His Ala Val Thr Phe Val Asp Asn His Asp SerGln Pro 325 330 335 gaa gaa gct tta gag tct ttt gtt gaa gaa tgg ttc aaacca tta gcg 1056 Glu Glu Ala Leu Glu Ser Phe Val Glu Glu Trp Phe Lys ProLeu Ala 340 345 350 tat gct ttg aca tta aca cgt gaa caa ggc tac cct tctgta ttt tat 1104 Tyr Ala Leu Thr Leu Thr Arg Glu Gln Gly Tyr Pro Ser ValPhe Tyr 355 360 365 gga gat tat tat ggc att cca acg cat ggt gta cca gcgatg aaa tcg 1152 Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala MetLys Ser 370 375 380 aaa att gac ccg att cta gaa gcg cgt caa aag tat gcatat gga aga 1200 Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala TyrGly Arg 385 390 395 400 caa aat gac tac tta gac cat cat aat atc atc ggttgg aca cgt gaa 1248 Gln Asn Asp Tyr Leu Asp His His Asn Ile Ile Gly TrpThr Arg Glu 405 410 415 ggg aat aca gca cac ccc aac tcc ggt tta gct actatc atg tcc gat 1296 Gly Asn Thr Ala His Pro Asn Ser Gly Leu Ala Thr IleMet Ser Asp 420 425 430 ggg gca gga gga aat aag tgg atg ttt gtt ggg cgtaat aaa gct ggt 1344 Gly Ala Gly Gly Asn Lys Trp Met Phe Val Gly Arg AsnLys Ala Gly 435 440 445 caa gtt tgg acc gat atc act gga aat cgt gca ggtact gtt acg att 1392 Gln Val Trp Thr Asp Ile Thr Gly Asn Arg Ala Gly ThrVal Thr Ile 450 455 460 aat gct gat gga tgg ggt aat ttt tct gta aat ggagga tca gtt tct 1440 Asn Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly GlySer Val Ser 465 470 475 480 att tgg gta aac aaa taa 1458 Ile Trp Val AsnLys 485 12 485 PRT Bacillus sp. 12 His His Asn Gly Thr Asn Gly Thr MetMet Gln Tyr Phe Glu Trp Tyr 1 5 10 15 Leu Pro Asn Asp Gly Asn His TrpAsn Arg Leu Arg Ser Asp Ala Ser 20 25 30 Asn Leu Lys Asp Lys Gly Ile SerAla Val Trp Ile Pro Pro Ala Trp 35 40 45 Lys Gly Ala Ser Gln Asn Asp ValGly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60 Asp Leu Gly Glu Phe Asn Gln LysGly Thr Ile Arg Thr Lys Tyr Gly 65 70 75 80 Thr Arg Asn Gln Leu Gln AlaAla Val Asn Ala Leu Lys Ser Asn Gly 85 90 95 Ile Gln Val Tyr Gly Asp ValVal Met Asn His Lys Gly Gly Ala Asp 100 105 110 Ala Thr Glu Met Val ArgAla Val Glu Val Asn Pro Asn Asn Arg Asn 115 120 125 Gln Glu Val Ser GlyGlu Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp 130 135 140 Phe Pro Gly ArgGly Asn Thr His Ser Asn Phe Lys Trp Arg Trp Tyr 145 150 155 160 His PheAsp Gly Val Asp Trp Asp Gln Ser Arg Lys Leu Asn Asn Arg 165 170 175 IleTyr Lys Phe Arg Gly Asp Gly Lys Gly Trp Asp Trp Glu Val Asp 180 185 190Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met 195 200205 Asp His Pro Glu Val Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr 210215 220 Thr Asn Thr Leu Gly Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His225 230 235 240 Ile Lys Tyr Ser Phe Thr Arg Asp Trp Ile Asn His Val ArgSer Ala 245 250 255 Thr Gly Lys Asn Met Phe Ala Val Ala Glu Phe Trp LysAsn Asp Leu 260 265 270 Gly Ala Ile Glu Asn Tyr Leu Asn Lys Thr Asn TrpAsn His Ser Val 275 280 285 Phe Asp Val Pro Leu His Tyr Asn Leu Tyr AsnAla Ser Lys Ser Gly 290 295 300 Gly Asn Tyr Asp Met Arg Gln Ile Phe AsnGly Thr Val Val Gln Arg 305 310 315 320 His Pro Met His Ala Val Thr PheVal Asp Asn His Asp Ser Gln Pro 325 330 335 Glu Glu Ala Leu Glu Ser PheVal Glu Glu Trp Phe Lys Pro Leu Ala 340 345 350 Tyr Ala Leu Thr Leu ThrArg Glu Gln Gly Tyr Pro Ser Val Phe Tyr 355 360 365 Gly Asp Tyr Tyr GlyIle Pro Thr His Gly Val Pro Ala Met Lys Ser 370 375 380 Lys Ile Asp ProIle Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly Arg 385 390 395 400 Gln AsnAsp Tyr Leu Asp His His Asn Ile Ile Gly Trp Thr Arg Glu 405 410 415 GlyAsn Thr Ala His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp 420 425 430Gly Ala Gly Gly Asn Lys Trp Met Phe Val Gly Arg Asn Lys Ala Gly 435 440445 Gln Val Trp Thr Asp Ile Thr Gly Asn Arg Ala Gly Thr Val Thr Ile 450455 460 Asn Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser465 470 475 480 Ile Trp Val Asn Lys 485 13 197 PRT Bacillus sp 707 13Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Lys Ser Gly 1 5 1015 Gly Asn Tyr Asp Met Arg Asn Ile Phe Asn Gly Thr Val Val Gln Arg 20 2530 His Pro Ser His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro 35 4045 Glu Glu Ala Leu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu Ala 50 5560 Tyr Ala Leu Thr Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr 65 7075 80 Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Arg Ser 8590 95 Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly Lys100 105 110 Gln Asn Asp Tyr Leu Asp His His Asn Ile Ile Gly Trp Thr ArgGlu 115 120 125 Gly Asn Thr Ala His Pro Asn Ser Gly Leu Ala Thr Ile MetSer Asp 130 135 140 Gly Ala Gly Gly Ser Lys Trp Met Phe Val Gly Arg AsnLys Ala Gly 145 150 155 160 Gln Val Trp Ser Asp Ile Thr Gly Asn Arg ThrGly Thr Val Thr Ile 165 170 175 Asn Ala Asp Gly Trp Gly Asn Phe Ser ValAsn Gly Gly Ser Val Ser 180 185 190 Ile Trp Val Asn Lys 195 14 24 DNAArtificial Sequence Primer 22149 14 cgattgctga cgctgttatt tgcg 24 15 19DNA Artificial Sequence Primer 24814 15 gatcacccgc gataccgtc 19 16 31DNA Artificial Sequence Primer # 24 16 gaatgtatgt cggccggcaa aacgccggtga 31 17 30 DNA Artificial Sequence Primer # 27 17 gccgccgctg ctgcagaatgaggcagcaag 30 18 48 DNA Artificial Sequence Primer # 312 18 cccgaaagctgaaccgcatc tataggtttc aagggaagac ttgggatt 48 19 23 DNA ArtificialSequence Primer 290 19 aggatggtca taatcaaagt cgg 23 20 52 DNA ArtificialSequence Primer #313 20 ccgactttga ttatgaccat cctgttgtcg tagcagagattaagagatgg gg 52 21 45 DNA Artificial Sequence Primer # 314 21cgacaatgtc atggtggtcg aaaaaatcat gctgtgctcc gtacg 45 22 23 DNAArtificial Sequence Primer #296 22 tttcgaccac catgacattg tcg 23 23 24DNA Artificial Sequence Primer #305 23 tatagatgcg gttcagcttt cggg 24 241650 DNA Bacillus sp. 24 cttgaatcat tatttaaagc tggttatgat atatgtaagcgttatcatta aaaggaggta 60 tttgatgaaa agatgggtag tagcaatgct ggcagtgttatttttatttc cttcggtagt 120 agttgcagat ggcttgaatg gaacgatgat gcagtattatgagtggcatc tagagaatga 180 tgggcaacac tggaatcggt tgcatgatga tgccgaagctttaagtaatg cgggtattac 240 agctatttgg atacccccag cctacaaagg aaatagtcaggctgatgttg ggtatggtgc 300 atacgacctt tatgatttag gggagtttaa tcaaaaaggtaccgttcgaa cgaaatacgg 360 gacaaaggct cagcttgagc gagctatagg gtccctaaagtcgaatgata tcaatgttta 420 tggggatgtc gtaatgaatc ataaattagg agctgatttcacggaggcag tgcaagctgt 480 tcaagtaaat ccttcgaacc gttggcagga tatttcaggtgtctacacga ttgatgcatg 540 gacgggattt gactttccag ggcgcaacaa tgcctattccgattttaaat ggagatggtt 600 ccattttaat ggcgttgact gggatcaacg ctatcaagaaaaccatcttt ttcgctttgc 660 aaatacgaac tggaactggc gagtggatga agagaatggtaattatgact atttattagg 720 atcgaacatt gactttagcc acccagaggt tcaagaggaattaaaggatt gggggagctg 780 gtttacggat gagctagatt tagatgggta tcgattggatgctattaagc atattccatt 840 ctggtatacg tcagattggg ttaggcatca gcgaagtgaagcagaccaag atttatttgt 900 cgtaggggag tattggaagg atgacgtagg tgctctcgaattttatttag atgaaatgaa 960 ttgggagatg tctctattcg atgttccgct caattataatttttaccggg cttcaaagca 1020 aggcggaagc tatgatatgc gtaatatttt acgaggatctttagtagaag cacatccgat 1080 tcatgcagtt acgtttgttg ataatcatga tactcagccaggagagtcat tagaatcatg 1140 ggtcgctgat tggtttaagc cacttgctta tgcgacaatcttgacgcgtg aaggtggtta 1200 tccaaatgta ttttacggtg actactatgg gattcctaacgataacattt cagctaagaa 1260 ggatatgatt gatgagttgc ttgatgcacg tcaaaattacgcatatggca cacaacatga 1320 ctattttgat cattgggata tcgttggatg gacaagagaaggtacatcct cacgtcctaa 1380 ttcgggtctt gctactatta tgtccaatgg tcctggaggatcaaaatgga tgtacgtagg 1440 acagcaacat gcaggacaaa cgtggacaga tttaactggcaatcacgcgg cgtcggttac 1500 gattaatggt gatggctggg gcgaattctt tacaaatggaggatctgtat ccgtgtatgt 1560 gaaccaataa taaaaagcct tgagaaggga ttcctccctaactcaaggct ttctttatgt 1620 cgtttagctc aacgcttcta cgaagcttta 1650 25 501PRT Bacillus sp. 25 Met Lys Arg Trp Val Val Ala Met Leu Ala Val Leu PheLeu Phe Pro 1 5 10 15 Ser Val Val Val Ala Asp Gly Leu Asn Gly Thr MetMet Gln Tyr Tyr 20 25 30 Glu Trp His Leu Glu Asn Asp Gly Gln His Trp AsnArg Leu His Asp 35 40 45 Asp Ala Glu Ala Leu Ser Asn Ala Gly Ile Thr AlaIle Trp Ile Pro 50 55 60 Pro Ala Tyr Lys Gly Asn Ser Gln Ala Asp Val GlyTyr Gly Ala Tyr 65 70 75 80 Asp Leu Tyr Asp Leu Gly Glu Phe Asn Gln LysGly Thr Val Arg Thr 85 90 95 Lys Tyr Gly Thr Lys Ala Gln Leu Glu Arg AlaIle Gly Ser Leu Lys 100 105 110 Ser Asn Asp Ile Asn Val Tyr Gly Asp ValVal Met Asn His Lys Leu 115 120 125 Gly Ala Asp Phe Thr Glu Ala Val GlnAla Val Gln Val Asn Pro Ser 130 135 140 Asn Arg Trp Gln Asp Ile Ser GlyVal Tyr Thr Ile Asp Ala Trp Thr 145 150 155 160 Gly Phe Asp Phe Pro GlyArg Asn Asn Ala Tyr Ser Asp Phe Lys Trp 165 170 175 Arg Trp Phe His PheAsn Gly Val Asp Trp Asp Gln Arg Tyr Gln Glu 180 185 190 Asn His Leu PheArg Phe Ala Asn Thr Asn Trp Asn Trp Arg Val Asp 195 200 205 Glu Glu AsnGly Asn Tyr Asp Tyr Leu Leu Gly Ser Asn Ile Asp Phe 210 215 220 Ser HisPro Glu Val Gln Glu Glu Leu Lys Asp Trp Gly Ser Trp Phe 225 230 235 240Thr Asp Glu Leu Asp Leu Asp Gly Tyr Arg Leu Asp Ala Ile Lys His 245 250255 Ile Pro Phe Trp Tyr Thr Ser Asp Trp Val Arg His Gln Arg Ser Glu 260265 270 Ala Asp Gln Asp Leu Phe Val Val Gly Glu Tyr Trp Lys Asp Asp Val275 280 285 Gly Ala Leu Glu Phe Tyr Leu Asp Glu Met Asn Trp Glu Met SerLeu 290 295 300 Phe Asp Val Pro Leu Asn Tyr Asn Phe Tyr Arg Ala Ser LysGln Gly 305 310 315 320 Gly Ser Tyr Asp Met Arg Asn Ile Leu Arg Gly SerLeu Val Glu Ala 325 330 335 His Pro Ile His Ala Val Thr Phe Val Asp AsnHis Asp Thr Gln Pro 340 345 350 Gly Glu Ser Leu Glu Ser Trp Val Ala AspTrp Phe Lys Pro Leu Ala 355 360 365 Tyr Ala Thr Ile Leu Thr Arg Glu GlyGly Tyr Pro Asn Val Phe Tyr 370 375 380 Gly Asp Tyr Tyr Gly Ile Pro AsnAsp Asn Ile Ser Ala Lys Lys Asp 385 390 395 400 Met Ile Asp Glu Leu LeuAsp Ala Arg Gln Asn Tyr Ala Tyr Gly Thr 405 410 415 Gln His Asp Tyr PheAsp His Trp Asp Ile Val Gly Trp Thr Arg Glu 420 425 430 Gly Thr Ser SerArg Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asn 435 440 445 Gly Pro GlyGly Ser Lys Trp Met Tyr Val Gly Gln Gln His Ala Gly 450 455 460 Gln ThrTrp Thr Asp Leu Thr Gly Asn His Ala Ala Ser Val Thr Ile 465 470 475 480Asn Gly Asp Gly Trp Gly Glu Phe Phe Thr Asn Gly Gly Ser Val Ser 485 490495 Val Tyr Val Asn Gln 500 26 1745 DNA Bacillus sp. CDS (190)..(1692)26 aactaagtaa catcgattca ggataaaagt atgcgaaacg atgcgcaaaa ctgcgcaact 60actagcactc ttcagggact aaaccacctt ttttccaaaa atgacatcat ataaacaaat 120ttgtctacca atcactattt aaagctgttt atgatatatg taagcgttat cattaaaagg 180aggtatttg atg aga aga tgg gta gta gca atg ttg gca gtg tta ttt tta 231Met Arg Arg Trp Val Val Ala Met Leu Ala Val Leu Phe Leu -20 -15 -10 tttcct tcg gta gta gtt gca gat gga ttg aac ggt acg atg atg cag 279 Phe ProSer Val Val Val Ala Asp Gly Leu Asn Gly Thr Met Met Gln -5 -1 1 5 tattat gag tgg cat ttg gaa aac gac ggg cag cat tgg aat cgg ttg 327 Tyr TyrGlu Trp His Leu Glu Asn Asp Gly Gln His Trp Asn Arg Leu 10 15 20 25 cacgat gat gcc gca gct ttg agt gat gct ggt att aca gct att tgg 375 His AspAsp Ala Ala Ala Leu Ser Asp Ala Gly Ile Thr Ala Ile Trp 30 35 40 att ccgcca gcc tac aaa ggt aat agt cag gcg gat gtt ggg tac ggt 423 Ile Pro ProAla Tyr Lys Gly Asn Ser Gln Ala Asp Val Gly Tyr Gly 45 50 55 gca tac gatctt tat gat tta gga gag ttc aat caa aag ggt act gtt 471 Ala Tyr Asp LeuTyr Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val 60 65 70 cga acg aaa tacgga act aag gca cag ctt gaa cga gct att ggg tcc 519 Arg Thr Lys Tyr GlyThr Lys Ala Gln Leu Glu Arg Ala Ile Gly Ser 75 80 85 ctt aaa tct aat gatatc aat gta tac gga gat gtc gtg atg aat cat 567 Leu Lys Ser Asn Asp IleAsn Val Tyr Gly Asp Val Val Met Asn His 90 95 100 105 aaa atg gga gctgat ttt acg gag gca gtg caa gct gtt caa gta aat 615 Lys Met Gly Ala AspPhe Thr Glu Ala Val Gln Ala Val Gln Val Asn 110 115 120 cca acg aat cgttgg cag gat att tca ggt gcc tac acg att gat gcg 663 Pro Thr Asn Arg TrpGln Asp Ile Ser Gly Ala Tyr Thr Ile Asp Ala 125 130 135 tgg acg ggt ttcgac ttt tca ggg cgt aac aac gcc tat tca gat ttt 711 Trp Thr Gly Phe AspPhe Ser Gly Arg Asn Asn Ala Tyr Ser Asp Phe 140 145 150 aag tgg aga tggttc cat ttt aat ggt gtt gac tgg gat cag cgc tat 759 Lys Trp Arg Trp PheHis Phe Asn Gly Val Asp Trp Asp Gln Arg Tyr 155 160 165 caa gaa aat catatt ttc cgc ttt gca aat acg aac tgg aac tgg cga 807 Gln Glu Asn His IlePhe Arg Phe Ala Asn Thr Asn Trp Asn Trp Arg 170 175 180 185 gtg gat gaagag aac ggt aat tat gat tac ctg tta gga tcg aat atc 855 Val Asp Glu GluAsn Gly Asn Tyr Asp Tyr Leu Leu Gly Ser Asn Ile 190 195 200 gac ttt agtcat cca gaa gta caa gat gag ttg aag gat tgg ggt agc 903 Asp Phe Ser HisPro Glu Val Gln Asp Glu Leu Lys Asp Trp Gly Ser 205 210 215 tgg ttt accgat gag tta gat ttg gat ggt tat cgt tta gat gct att 951 Trp Phe Thr AspGlu Leu Asp Leu Asp Gly Tyr Arg Leu Asp Ala Ile 220 225 230 aaa cat attcca ttc tgg tat aca tct gat tgg gtt cgg cat cag cgc 999 Lys His Ile ProPhe Trp Tyr Thr Ser Asp Trp Val Arg His Gln Arg 235 240 245 aac gaa gcagat caa gat tta ttt gtc gta ggg gaa tat tgg aag gat 1047 Asn Glu Ala AspGln Asp Leu Phe Val Val Gly Glu Tyr Trp Lys Asp 250 255 260 265 gac gtaggt gct ctc gaa ttt tat tta gat gaa atg aat tgg gag atg 1095 Asp Val GlyAla Leu Glu Phe Tyr Leu Asp Glu Met Asn Trp Glu Met 270 275 280 tct ctattc gat gtt cca ctt aat tat aat ttt tac cgg gct tca caa 1143 Ser Leu PheAsp Val Pro Leu Asn Tyr Asn Phe Tyr Arg Ala Ser Gln 285 290 295 caa ggtgga agc tat gat atg cgt aat att tta cga gga tct tta gta 1191 Gln Gly GlySer Tyr Asp Met Arg Asn Ile Leu Arg Gly Ser Leu Val 300 305 310 gaa gcgcat ccg atg cat gca gtt acg ttt gtt gat aat cat gat act 1239 Glu Ala HisPro Met His Ala Val Thr Phe Val Asp Asn His Asp Thr 315 320 325 cag ccaggg gag tca tta gag tca tgg gtt gct gat tgg ttt aag cca 1287 Gln Pro GlyGlu Ser Leu Glu Ser Trp Val Ala Asp Trp Phe Lys Pro 330 335 340 345 cttgct tat gcg aca att ttg acg cgt gaa ggt ggt tat cca aat gta 1335 Leu AlaTyr Ala Thr Ile Leu Thr Arg Glu Gly Gly Tyr Pro Asn Val 350 355 360 ttttac ggt gat tac tat ggg att cct aac gat aac att tca gct aaa 1383 Phe TyrGly Asp Tyr Tyr Gly Ile Pro Asn Asp Asn Ile Ser Ala Lys 365 370 375 aaagat atg att gat gag ctg ctt gat gca cgt caa aat tac gca tat 1431 Lys AspMet Ile Asp Glu Leu Leu Asp Ala Arg Gln Asn Tyr Ala Tyr 380 385 390 ggcacg cag cat gac tat ttt gat cat tgg gat gtt gta gga tgg act 1479 Gly ThrGln His Asp Tyr Phe Asp His Trp Asp Val Val Gly Trp Thr 395 400 405 agggaa gga tct tcc tcc aga cct aat tca ggc ctt gcg act att atg 1527 Arg GluGly Ser Ser Ser Arg Pro Asn Ser Gly Leu Ala Thr Ile Met 410 415 420 425tcg aat gga cct ggt ggt tcc aag tgg atg tat gta gga cgt cag aat 1575 SerAsn Gly Pro Gly Gly Ser Lys Trp Met Tyr Val Gly Arg Gln Asn 430 435 440gca gga caa aca tgg aca gat tta act ggt aat aac gga gcg tcc gtt 1623 AlaGly Gln Thr Trp Thr Asp Leu Thr Gly Asn Asn Gly Ala Ser Val 445 450 455aca att aat ggc gat gga tgg ggc gaa ttc ttt acg aat gga gga tct 1671 ThrIle Asn Gly Asp Gly Trp Gly Glu Phe Phe Thr Asn Gly Gly Ser 460 465 470gta tcc gtg tac gtg aac caa taacaaaaag ccttgagaag ggattcctcc 1722 ValSer Val Tyr Val Asn Gln 475 480 ctaactcaag gctttcttta tgt 1745 27 501PRT Bacillus sp. 27 Met Arg Arg Trp Val Val Ala Met Leu Ala Val Leu PheLeu Phe Pro -20 -15 -10 Ser Val Val Val Ala Asp Gly Leu Asn Gly Thr MetMet Gln Tyr Tyr -5 -1 1 5 10 Glu Trp His Leu Glu Asn Asp Gly Gln His TrpAsn Arg Leu His Asp 15 20 25 Asp Ala Ala Ala Leu Ser Asp Ala Gly Ile ThrAla Ile Trp Ile Pro 30 35 40 Pro Ala Tyr Lys Gly Asn Ser Gln Ala Asp ValGly Tyr Gly Ala Tyr 45 50 55 Asp Leu Tyr Asp Leu Gly Glu Phe Asn Gln LysGly Thr Val Arg Thr 60 65 70 75 Lys Tyr Gly Thr Lys Ala Gln Leu Glu ArgAla Ile Gly Ser Leu Lys 80 85 90 Ser Asn Asp Ile Asn Val Tyr Gly Asp ValVal Met Asn His Lys Met 95 100 105 Gly Ala Asp Phe Thr Glu Ala Val GlnAla Val Gln Val Asn Pro Thr 110 115 120 Asn Arg Trp Gln Asp Ile Ser GlyAla Tyr Thr Ile Asp Ala Trp Thr 125 130 135 Gly Phe Asp Phe Ser Gly ArgAsn Asn Ala Tyr Ser Asp Phe Lys Trp 140 145 150 155 Arg Trp Phe His PheAsn Gly Val Asp Trp Asp Gln Arg Tyr Gln Glu 160 165 170 Asn His Ile PheArg Phe Ala Asn Thr Asn Trp Asn Trp Arg Val Asp 175 180 185 Glu Glu AsnGly Asn Tyr Asp Tyr Leu Leu Gly Ser Asn Ile Asp Phe 190 195 200 Ser HisPro Glu Val Gln Asp Glu Leu Lys Asp Trp Gly Ser Trp Phe 205 210 215 ThrAsp Glu Leu Asp Leu Asp Gly Tyr Arg Leu Asp Ala Ile Lys His 220 225 230235 Ile Pro Phe Trp Tyr Thr Ser Asp Trp Val Arg His Gln Arg Asn Glu 240245 250 Ala Asp Gln Asp Leu Phe Val Val Gly Glu Tyr Trp Lys Asp Asp Val255 260 265 Gly Ala Leu Glu Phe Tyr Leu Asp Glu Met Asn Trp Glu Met SerLeu 270 275 280 Phe Asp Val Pro Leu Asn Tyr Asn Phe Tyr Arg Ala Ser GlnGln Gly 285 290 295 Gly Ser Tyr Asp Met Arg Asn Ile Leu Arg Gly Ser LeuVal Glu Ala 300 305 310 315 His Pro Met His Ala Val Thr Phe Val Asp AsnHis Asp Thr Gln Pro 320 325 330 Gly Glu Ser Leu Glu Ser Trp Val Ala AspTrp Phe Lys Pro Leu Ala 335 340 345 Tyr Ala Thr Ile Leu Thr Arg Glu GlyGly Tyr Pro Asn Val Phe Tyr 350 355 360 Gly Asp Tyr Tyr Gly Ile Pro AsnAsp Asn Ile Ser Ala Lys Lys Asp 365 370 375 Met Ile Asp Glu Leu Leu AspAla Arg Gln Asn Tyr Ala Tyr Gly Thr 380 385 390 395 Gln His Asp Tyr PheAsp His Trp Asp Val Val Gly Trp Thr Arg Glu 400 405 410 Gly Ser Ser SerArg Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asn 415 420 425 Gly Pro GlyGly Ser Lys Trp Met Tyr Val Gly Arg Gln Asn Ala Gly 430 435 440 Gln ThrTrp Thr Asp Leu Thr Gly Asn Asn Gly Ala Ser Val Thr Ile 445 450 455 AsnGly Asp Gly Trp Gly Glu Phe Phe Thr Asn Gly Gly Ser Val Ser 460 465 470475 Val Tyr Val Asn Gln 480 28 501 PRT Bacillus sp 28 Met Arg Arg TrpVal Val Ala Met Leu Ala Val Leu Phe Leu Phe Pro 1 5 10 15 Ser Val ValVal Ala Asp Gly Leu Asn Gly Thr Met Met Gln Tyr Tyr 20 25 30 Glu Trp HisLeu Glu Asn Asp Gly Gln His Trp Asn Arg Leu His Asp 35 40 45 Asp Ala AlaAla Leu Ser Asp Ala Gly Ile Thr Ala Ile Trp Ile Pro 50 55 60 Pro Ala TyrLys Gly Asn Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr 65 70 75 80 Asp LeuTyr Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr 85 90 95 Lys TyrGly Thr Lys Ala Gln Leu Glu Arg Ala Ile Gly Ser Leu Lys 100 105 110 SerAsn Asp Ile Asn Val Tyr Gly Asp Val Val Met Asn His Lys Met 115 120 125Gly Ala Asp Phe Thr Glu Ala Val Gln Ala Val Gln Val Asn Pro Thr 130 135140 Asn Arg Trp Gln Asp Ile Ser Gly Ala Tyr Thr Ile Asp Ala Trp Thr 145150 155 160 Gly Phe Asp Phe Ser Gly Arg Asn Asn Ala Tyr Ser Asp Phe LysTrp 165 170 175 Arg Trp Phe His Phe Asn Gly Val Asp Trp Asp Gln Arg TyrGln Glu 180 185 190 Asn His Ile Phe Arg Phe Ala Asn Thr Asn Trp Asn TrpArg Val Asp 195 200 205 Glu Glu Asn Gly Asn Tyr Asp Tyr Leu Leu Gly SerAsn Ile Asp Phe 210 215 220 Ser His Pro Glu Val Gln Asp Glu Leu Lys AspTrp Gly Ser Trp Phe 225 230 235 240 Thr Asp Glu Leu Asp Leu Asp Gly TyrArg Leu Asp Ala Ile Lys His 245 250 255 Ile Pro Phe Trp Tyr Thr Ser AspTrp Val Arg His Gln Arg Asn Glu 260 265 270 Ala Asp Gln Asp Leu Phe ValVal Gly Glu Tyr Trp Lys Asp Asp Val 275 280 285 Gly Ala Leu Glu Phe TyrLeu Asp Glu Met Asn Trp Glu Met Ser Leu 290 295 300 Phe Asp Val Pro LeuAsn Tyr Asn Phe Tyr Arg Ala Ser Gln Gln Gly 305 310 315 320 Gly Ser TyrAsp Met Arg Asn Ile Leu Arg Gly Ser Leu Val Glu Ala 325 330 335 His ProMet His Ala Val Thr Phe Val Asp Asn His Asp Thr Gln Pro 340 345 350 GlyGlu Ser Leu Glu Ser Trp Val Ala Asp Trp Phe Lys Pro Leu Ala 355 360 365Tyr Ala Thr Ile Leu Thr Arg Glu Gly Gly Tyr Pro Asn Val Phe Tyr 370 375380 Gly Asp Tyr Tyr Gly Ile Pro Asn Asp Asn Ile Ser Ala Lys Lys Asp 385390 395 400 Met Ile Asp Glu Leu Leu Asp Ala Arg Gln Asn Tyr Ala Tyr GlyThr 405 410 415 Gln His Asp Tyr Phe Asp His Trp Asp Val Val Gly Trp ThrArg Glu 420 425 430 Gly Ser Ser Ser Arg Pro Asn Ser Gly Leu Ala Thr IleMet Ser Asn 435 440 445 Gly Pro Gly Gly Ser Lys Trp Met Tyr Val Gly ArgGln Asn Ala Gly 450 455 460 Gln Thr Trp Thr Asp Leu Thr Gly Asn Asn GlyAla Ser Val Thr Ile 465 470 475 480 Asn Gly Asp Gly Trp Gly Glu Phe PheThr Asn Gly Gly Ser Val Ser 485 490 495 Val Tyr Val Asn Gln 500 29 1920DNA Bacillus licheniformis CDS (421)..(1872) 29 cggaagattg gaagtacaaaaataagcaaa agattgtcaa tcatgtcatg agccatgcgg 60 gagacggaaa aatcgtcttaatgcacgata tttatgcaac gttcgcagat gctgctgaag 120 agattattaa aaagctgaaagcaaaaggct atcaattggt aactgtatct cagcttgaag 180 aagtgaagaa gcagagaggctattgaataa atgagtagaa gcgccatatc ggcgcttttc 240 ttttggaaga aaatatagggaaaatggtac ttgttaaaaa ttcggaatat ttatacaaca 300 tcatatgttt cacattgaaaggggaggaga atcatgaaac aacaaaaacg gctttacgcc 360 cgattgctga cgctgttatttgcgctcatc ttcttgctgc ctcattctgc agcagcggcg 420 gca aat ctt aat ggg acgctg atg cag tat ttt gaa tgg tac atg ccc 468 Ala Asn Leu Asn Gly Thr LeuMet Gln Tyr Phe Glu Trp Tyr Met Pro 1 5 10 15 aat gac ggc caa cat tggagg cgt ttg caa aac gac tcg gca tat ttg 516 Asn Asp Gly Gln His Trp ArgArg Leu Gln Asn Asp Ser Ala Tyr Leu 20 25 30 gct gaa cac ggt att act gccgtc tgg att ccc ccg gca tat aag gga 564 Ala Glu His Gly Ile Thr Ala ValTrp Ile Pro Pro Ala Tyr Lys Gly 35 40 45 acg agc caa gcg gat gtg ggc tacggt gct tac gac ctt tat gat tta 612 Thr Ser Gln Ala Asp Val Gly Tyr GlyAla Tyr Asp Leu Tyr Asp Leu 50 55 60 ggg gag ttt cat caa aaa ggg acg gttcgg aca aag tac ggc aca aaa 660 Gly Glu Phe His Gln Lys Gly Thr Val ArgThr Lys Tyr Gly Thr Lys 65 70 75 80 gga gag ctg caa tct gcg atc aaa agtctt cat tcc cgc gac att aac 708 Gly Glu Leu Gln Ser Ala Ile Lys Ser LeuHis Ser Arg Asp Ile Asn 85 90 95 gtt tac ggg gat gtg gtc atc aac cac aaaggc ggc gct gat gcg acc 756 Val Tyr Gly Asp Val Val Ile Asn His Lys GlyGly Ala Asp Ala Thr 100 105 110 gaa gat gta acc gcg gtt gaa gtc gat cccgct gac cgc aac cgc gta 804 Glu Asp Val Thr Ala Val Glu Val Asp Pro AlaAsp Arg Asn Arg Val 115 120 125 att tca gga gaa cac cta att aaa gcc tggaca cat ttt cat ttt ccg 852 Ile Ser Gly Glu His Leu Ile Lys Ala Trp ThrHis Phe His Phe Pro 130 135 140 ggg cgc ggc agc aca tac agc gat ttt aaatgg cat tgg tac cat ttt 900 Gly Arg Gly Ser Thr Tyr Ser Asp Phe Lys TrpHis Trp Tyr His Phe 145 150 155 160 gac gga acc gat tgg gac gag tcc cgaaag ctg aac cgc atc tat aag 948 Asp Gly Thr Asp Trp Asp Glu Ser Arg LysLeu Asn Arg Ile Tyr Lys 165 170 175 ttt caa gga aag gct tgg gat tgg gaagtt tcc aat gaa aac ggc aac 996 Phe Gln Gly Lys Ala Trp Asp Trp Glu ValSer Asn Glu Asn Gly Asn 180 185 190 tat gat tat ttg atg tat gcc gac atcgat tat gac cat cct gat gtc 1044 Tyr Asp Tyr Leu Met Tyr Ala Asp Ile AspTyr Asp His Pro Asp Val 195 200 205 gca gca gaa att aag aga tgg ggc acttgg tat gcc aat gaa ctg caa 1092 Ala Ala Glu Ile Lys Arg Trp Gly Thr TrpTyr Ala Asn Glu Leu Gln 210 215 220 ttg gac ggt ttc cgt ctt gat gct gtcaaa cac att aaa ttt tct ttt 1140 Leu Asp Gly Phe Arg Leu Asp Ala Val LysHis Ile Lys Phe Ser Phe 225 230 235 240 ttg cgg gat tgg gtt aat cat gtcagg gaa aaa acg ggg aag gaa atg 1188 Leu Arg Asp Trp Val Asn His Val ArgGlu Lys Thr Gly Lys Glu Met 245 250 255 ttt acg gta gct gaa tat tgg cagaat gac ttg ggc gcg ctg gaa aac 1236 Phe Thr Val Ala Glu Tyr Trp Gln AsnAsp Leu Gly Ala Leu Glu Asn 260 265 270 tat ttg aac aaa aca aat ttt aatcat tca gtg ttt gac gtg ccg ctt 1284 Tyr Leu Asn Lys Thr Asn Phe Asn HisSer Val Phe Asp Val Pro Leu 275 280 285 cat tat cag ttc cat gct gca tcgaca cag gga ggc ggc tat gat atg 1332 His Tyr Gln Phe His Ala Ala Ser ThrGln Gly Gly Gly Tyr Asp Met 290 295 300 agg aaa ttg ctg aac ggt acg gtcgtt tcc aag cat ccg ttg aaa tcg 1380 Arg Lys Leu Leu Asn Gly Thr Val ValSer Lys His Pro Leu Lys Ser 305 310 315 320 gtt aca ttt gtc gat aac catgat aca cag ccg ggg caa tcg ctt gag 1428 Val Thr Phe Val Asp Asn His AspThr Gln Pro Gly Gln Ser Leu Glu 325 330 335 tcg act gtc caa aca tgg tttaag ccg ctt gct tac gct ttt att ctc 1476 Ser Thr Val Gln Thr Trp Phe LysPro Leu Ala Tyr Ala Phe Ile Leu 340 345 350 aca agg gaa tct gga tac cctcag gtt ttc tac ggg gat atg tac ggg 1524 Thr Arg Glu Ser Gly Tyr Pro GlnVal Phe Tyr Gly Asp Met Tyr Gly 355 360 365 acg aaa gga gac tcc cag cgcgaa att cct gcc ttg aaa cac aaa att 1572 Thr Lys Gly Asp Ser Gln Arg GluIle Pro Ala Leu Lys His Lys Ile 370 375 380 gaa ccg atc tta aaa gcg agaaaa cag tat gcg tac gga gca cag cat 1620 Glu Pro Ile Leu Lys Ala Arg LysGln Tyr Ala Tyr Gly Ala Gln His 385 390 395 400 gat tat ttc gac cac catgac att gtc ggc tgg aca agg gaa ggc gac 1668 Asp Tyr Phe Asp His His AspIle Val Gly Trp Thr Arg Glu Gly Asp 405 410 415 agc tcg gtt gca aat tcaggt ttg gcg gca tta ata aca gac gga ccc 1716 Ser Ser Val Ala Asn Ser GlyLeu Ala Ala Leu Ile Thr Asp Gly Pro 420 425 430 ggt ggg gca aag cga atgtat gtc ggc cgg caa aac gcc ggt gag aca 1764 Gly Gly Ala Lys Arg Met TyrVal Gly Arg Gln Asn Ala Gly Glu Thr 435 440 445 tgg cat gac att acc ggaaac cgt tcg gag ccg gtt gtc atc aat tcg 1812 Trp His Asp Ile Thr Gly AsnArg Ser Glu Pro Val Val Ile Asn Ser 450 455 460 gaa ggc tgg gga gag tttcac gta aac ggc ggg tcg gtt tca att tat 1860 Glu Gly Trp Gly Glu Phe HisVal Asn Gly Gly Ser Val Ser Ile Tyr 465 470 475 480 gtt caa aga tagaagagcagag aggacggatt tcctgaagga aatccgtttt 1912 Val Gln Arg tttatttt1920 30 483 PRT Bacillus licheniformis 30 Ala Asn Leu Asn Gly Thr LeuMet Gln Tyr Phe Glu Trp Tyr Met Pro 1 5 10 15 Asn Asp Gly Gln His TrpArg Arg Leu Gln Asn Asp Ser Ala Tyr Leu 20 25 30 Ala Glu His Gly Ile ThrAla Val Trp Ile Pro Pro Ala Tyr Lys Gly 35 40 45 Thr Ser Gln Ala Asp ValGly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu 50 55 60 Gly Glu Phe His Gln LysGly Thr Val Arg Thr Lys Tyr Gly Thr Lys 65 70 75 80 Gly Glu Leu Gln SerAla Ile Lys Ser Leu His Ser Arg Asp Ile Asn 85 90 95 Val Tyr Gly Asp ValVal Ile Asn His Lys Gly Gly Ala Asp Ala Thr 100 105 110 Glu Asp Val ThrAla Val Glu Val Asp Pro Ala Asp Arg Asn Arg Val 115 120 125 Ile Ser GlyGlu His Leu Ile Lys Ala Trp Thr His Phe His Phe Pro 130 135 140 Gly ArgGly Ser Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe 145 150 155 160Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys 165 170175 Phe Gln Gly Lys Ala Trp Asp Trp Glu Val Ser Asn Glu Asn Gly Asn 180185 190 Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Tyr Asp His Pro Asp Val195 200 205 Ala Ala Glu Ile Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu LeuGln 210 215 220 Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys PheSer Phe 225 230 235 240 Leu Arg Asp Trp Val Asn His Val Arg Glu Lys ThrGly Lys Glu Met 245 250 255 Phe Thr Val Ala Glu Tyr Trp Gln Asn Asp LeuGly Ala Leu Glu Asn 260 265 270 Tyr Leu Asn Lys Thr Asn Phe Asn His SerVal Phe Asp Val Pro Leu 275 280 285 His Tyr Gln Phe His Ala Ala Ser ThrGln Gly Gly Gly Tyr Asp Met 290 295 300 Arg Lys Leu Leu Asn Gly Thr ValVal Ser Lys His Pro Leu Lys Ser 305 310 315 320 Val Thr Phe Val Asp AsnHis Asp Thr Gln Pro Gly Gln Ser Leu Glu 325 330 335 Ser Thr Val Gln ThrTrp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu 340 345 350 Thr Arg Glu SerGly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly 355 360 365 Thr Lys GlyAsp Ser Gln Arg Glu Ile Pro Ala Leu Lys His Lys Ile 370 375 380 Glu ProIle Leu Lys Ala Arg Lys Gln Tyr Ala Tyr Gly Ala Gln His 385 390 395 400Asp Tyr Phe Asp His His Asp Ile Val Gly Trp Thr Arg Glu Gly Asp 405 410415 Ser Ser Val Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro 420425 430 Gly Gly Ala Lys Arg Met Tyr Val Gly Arg Gln Asn Ala Gly Glu Thr435 440 445 Trp His Asp Ile Thr Gly Asn Arg Ser Glu Pro Val Val Ile AsnSer 450 455 460 Glu Gly Trp Gly Glu Phe His Val Asn Gly Gly Ser Val SerIle Tyr 465 470 475 480 Val Gln Arg

1. A variant of an alpha-amylase having at least 60% homology to SEQ IDNO.8, comprising an alteration at one or more positions selected fromthe group of: 49, 60, 104, 132, 161, 170, 176, 179, 180, 181, 183, 200,203, 204, 207, 212, 237, 239, 250, 280, 298, 318, 374, 385, 393, 402,406, 427, 430, 440, 444, 447, 482, wherein (a) the alteration(s) areindependently (i) an insertion of an amino acid downstream of the aminoacid which occupies the position, (ii) a deletion of the amino acidwhich occupies the position, or (iii) a substitution of the amino acidwhich occupies the position with a different amino acid, (b) the varianthas alpha-amylase activity, and (c) each position corresponds to aposition of the amino acid sequence of the alpha-amylase having theamino acid sequence shown in SEQ ID NO: 8:
 2. The variant of claim 1,which variant has one or more of the following mutations: T49I; D60N;N104D; E132A,V,P; D161N; K170Q; K176R; G179N; K180T; A181N; D183N;D200N; X203Y; D204S; D207V,E,L,G; X212I; K237P; S239W; E250G,F; N280S;X298Q; L318M; Q374R; E385V; Q393R; Y402F; H406L,W; L427I D430N; V440A;N444R,K; E447Q,K; Q482K using SEQ ID NO: 8 for the numbering.
 3. Thevariant of claim 1 or 2, wherein the variant has the followingmutations: K170Q+D207V+N280S; E132A+D207V; D207E+E250G+H406L+L427I;D207V+L318M; D60N+D207V+L318M; T49I+E132V+V440A; T49I+K176R+D207V+Y402F;Q374R+E385V+Q393R; N190F+A209V+Q264S; G48A+T49I+G107A+I201F;T49I+G107A+I201F; G48A+T49I+I201F; G48A+T49I+G107A; T49I+I201F;T49I+G107A; G48A+T49I;N104D+D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+N444K+E447Q+Q482K;D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+N444K+E447Q+Q482K;D161N+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+N444K+E447Q+Q482K;D161N+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+E447Q+Q482K;N104D+D161lN+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+E447Q+Q482K;D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+E447Q+Q482K;N104D+D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N;D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N;H406W+D430N; N444K+E447Q+Q482K; E447Q+Q482K;N104D+D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+N444R+N444K+E447K+Q482K;D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+N444R+N444K+E447K+Q482K;N104D+D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W;D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W; H406W+D430N;N444K+E447K+Q482K; E447K+Q482K;N104D+D161N+A181N+D183N+D200N+D204S+K237P+S239W;N104D+D161N+A181N+D183N+D200N+D204S+K237P;N104D+D161N+A181N+D183N+D200N+D204S;D161N+A181N+D183N+D200N+D204S+K237P+S239W;D161N+A181N+D183N+D200N+D204S+K237P; D161N+A181N+D183N+D200N+D204S;K237P+S239W, using SEQ ID NO: 8 for the numbering.
 4. The variant of anyof claims 1-3, wherein the parent alpha-amylase is derived from a strainof B. licheniformis (SEQ ID NO: 8), B. amyloliquefaciens (SEQ ID NO:10), or B. stearothermophilus (SEQ ID NO: 6).
 5. The variant of any ofclaims 1-4, wherein the parent alpha-amylase is any of: LE174;LE174+G48A+T49I+G107A+I201F; LE174+M197L;LE174+G48A+T49I+G107A+M197L+I201F.
 6. The variant of claim 1, whereinthe variant is mutated in one or more of the following positions: T51I;D62N; N106D; D134A,V,P; D163N; X172Q; K179R; G184N; K185T; A186N; D188N;D205N; M208Y; D209S; X212V,E,L,G; L217I, K242P, S244W, N255G,F, N285S,S303Q, X323M; D387V, N395R; Y404F; H408L,W; X429I; D432N; V442A;X446R,K; X449Q,K; X484K, using SEQ ID NO: 4 for the numbering.
 7. Thevariant of claim 1 or 6, wherein the variant has the followingmutations: E212V+N285S; D134A+E212V; 255G+H408L+X429I; E212V+X323M;D62N+E212V+X323M; T51I+D134V+V442A; T51I+K179R+E212V+Y404F; D387V+N395R;N195F+X212V+K269S, when using SEQ ID NO: 4 for the numbering.
 8. Thevariant of any of claims 1-7, wherein the parent alpha-amylase isselected from the group comprising: SEQ ID NO: 2; SEQ ID NO: 4; SEQ IDNO: 12; SEQ ID NO: 13; or KSM-AP1378.
 9. The variant of any of claims1-8, wherein the parent alpha amylase is any of: SEQ ID NO.4+D183*+G184*; SEQ ID NO. 4+D183*+G184*+N195F; SP722+D183*+G184*+M202L;SEQ ID NO. 4+D183*+G184*+N195F+M202L; SEQ ID NO.6+I181*+G182*; SEQ IDNO.6+I181*+G182*+N193F; SEQ ID NO.6+I181*+G182*+M200L; SEQ IDNO.6+I181*+G182*+N193F+M200L; SEQ ID NO.12+D183*+G184*; SEQ IDNO.12+D183*+G184*+N195F; SEQ ID NO.12+D183*+G184*+M202L; SEQ IDNO.12+D183*+G184*+N195F+M202L.
 10. The variant of any of claims 1-9,wherein the parent alpha-amylase has an amino acid sequence which has adegree of identity to SEQ ID NO: 8 of at least 70%, more preferably atleast 80%, even more preferably at least about 90%, even more preferablyat least 95%, even more preferably at least 97%, and even morepreferably at least 99%.
 11. The variant of any of claims 1-10, whereinthe parent alpha-amylase is encoded by a nucleic acid sequence, whichhybridizes under low, preferably medium, preferred high stringencyconditions, with the nucleic acid sequence of SEQ ID NO:
 7. 12. Thevariant of any of claims 1-11, which variant has altered stability, inparticular at high temperatures from 70-120° C. and/or low pH in therange from pH 4-6.
 13. A DNA construct comprising a DNA sequenceencoding an alpha-amylase variant according to any one of claims 1-12.14. A recombinant expression vector which carries a DNA constructaccording to claim
 13. 15. A cell which is transformed with a DNAconstruct according to claim 13 or a vector according to claim
 14. 16.The cell according to claim 15, which is a microorganism, preferably abacterium or a fungus.
 17. The cell according to claim 16, which cell isa gram-positive bacterium, such as Bacillus subtilis, Bacilluslicheniformis, Bacillus lentus, Bacillus brevis, Bacillusstearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens,Bacillus coagulans, Bacillus circulans, Bacillus lautus or Bacillusthuringiensis.
 18. A composition comprising an alpha-amylase variant ofany of claims 1-12.
 19. The composition of claim 18, further comprisinga B. stearothermophilus alpha-amylase, particular in a ratio of 1:10 to10:1, preferably 1:2.
 20. The composition of claim 18 or 19, wherein thecomposition further comprises a glucoamylase, pullulanase and/or aphytase.
 21. A detergent composition comprising an alpha-amylase variantaccording to any of claims 1-12.
 22. A detergent composition of claim21, which additionally comprises another enzyme such as a protease, alipase, a peroxidase, another amylolytic enzyme, glucoamylase,maltogenic amylase, CGTase, mannanase, cutinase, laccase and/or acellulase.
 23. Use of an alpha-amylase variant according to any ofclaims 1-12 or a composition according to any of claims 18-20 for starchliquefaction.
 24. Use of an alpha-amylase variant according to any ofclaims 1-12 or a composition according to claims 18-20 for ethanolproduction.
 25. Use of an alpha-amylase variant according to any one ofclaims 1-12 or a composition according to claims 18-20 for washingand/or dishwashing.
 26. Use of an alpha-amylase variant of any one ofclaims 1-12 or a composition according to claims 18-20 for textiledesizing.